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Applications in High Resolution Mass Spectrometry: Food Safety and Pesticide Residue Analysis 1St Edition Romero-Gonz Lez - Ebook PDF
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Applications in High
Resolution Mass
Spectrometry
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Applications in High
Resolution Mass
Spectrometry
Food Safety and Pesticide
Residue Analysis
Edited by
Roberto Romero-González
To the fullest extent of the law, neither the Publisher nor the authors, contributors, or
editors, assume any liability for any injury and/or damage to persons or property as a
matter of products liability, negligence or otherwise, or from any use or operation of any
methods, products, instructions, or ideas contained in the material herein.
ISBN: 978-0-12-809464-8
v
vi Contents
Index...................................................................................................337
List of Contributors
Martha B. Adaime
Federal University of Santa Maria, Santa Maria, Brazil
Ana Agüera
CIESOL, Joint Centre of the University of Almerı́a e CIEMAT, Almerı́a, Spain
Franciso Javier Arrebola-Liébanas
University of Almerı́a, Almerı́a, Spain
Gabrieli Bernardi
Federal University of Santa Maria, Santa Maria, Brazil
Helen Botitsi
General Chemical State Laboratory, Athens, Greece
Jennifer A. Burgess
Waters Corporation, Milford, MA, United States
Marina Celia Campos-Mañas
CIESOL, Joint Centre of the University of Almerı́a e CIEMAT, Almerı́a, Spain
Gareth Cleland
Waters Corporation, Milford, MA, United States
Anastasios Economou
National and Kapodistrian University of Athens, Athens, Greece
Imma Ferrer
University of Colorado, Boulder, CO, United States
Antonia Garrido Frenich
University of Almerı́a, Almerı́a, Spain
M.T. Galceran
University of Barcelona, Barcelona, Spain
Juan F. Garcı́a-Reyes
University of Jaén, Jaén, Spain
Bienvenida Gilbert-López
University of Jaén, Jaén, Spain
Ana Belén Martı́nez-Piernas
CIESOL, Joint Centre of the University of Almerı́a e CIEMAT, Almerı́a, Spain
Antonio Molina Dı́az
University of Jaén, Jaén, Spain
xi
xii List of Contributors
David Moreno-González
University of Jaén, Jaén, Spain
E. Moyano
University of Barcelona, Barcelona, Spain
Lauren Mullin
Waters Corporation, Milford, MA, United States
Rocı́o Nortes-Méndez
University of Jaén, Jaén, Spain
P. Sivaperumal
National Institute of Occupational Health, Ahmedabad, Gujarat, India
Osmar D. Prestes
Federal University of Santa Maria, Santa Maria, Brazil
Roberto Romero-González
University of Almerı́a, Almerı́a, Spain
E. Michael Thurman
University of Colorado, Boulder, CO, United States
Despina Tsipi
General Chemical State Laboratory, Athens, Greece
Renato Zanella
Federal University of Santa Maria, Santa Maria, Brazil
Jerry A. Zweigenbaum
Agilent Technologies Inc., Wilmington, DE, United States
Preface
Each problem that I solved became a rule, which served afterward to solve other
problems.
Rene Descartes
Currently, mass spectrometry is an essential tool in food safety and its output has
reached an unprecedented level, being the most adequate detection system for the
analysis of pesticide residues in food matrices. In the last few years, new analyzers,
ionization methods, and analytical strategies have been developed to increase the
scope of analytical methods as well as the reliability of the results.
Up to now, high-resolution mass spectrometry (HRMS) has been considered as a
complementary tool of conventional triple quadrupole (QqQ) analyzers. However,
time of flight and/or Orbitrap are replacing low-resolution mass spectrometry ana-
lyzers because they can increase the number of compounds simultaneously moni-
tored, retrospective analysis can be carried out, and unknown compounds can be
identified. All these tasks can be performed using one benchtop platform at suitable
sensitivity. In addition, if HRMS analyzers are coupled with ultrahigh-performance
liquid chromatography (UHPLC) the number of compounds analyzed in one single
run can increase considerably. Furthermore, when ambient ionization techniques are
used with HRMS analyzers, sample treatment could be minimized, increasing sam-
ple throughput.
The use of these HRMS analyzers opens a new scenario in pesticide residue anal-
ysis, and potential users should know the strategies that can be applied to get all the
information that these analyzers could provide, as well as the pros and cons in rela-
tion to QqQ.
Because of the fast application and implementation of these new procedures, we
consider that setting the main principles and strategies based on these approaches is
necessary to provide a comprehensive view of the cited tools, being a cornerstone in
the food safety field.
Thus, this book would provide a complete overview of the possibilities that
HRMS could offer in pesticide residue analysis in food, as well as the suitable work-
flow needed to achieve the goals proposed by scientists. Chapters 1e4 provide an
overview of HRMS, basic concepts, hardware, general approaches (target and
nontarget analysis), and current legislation related to this topic. Chapters 5e7 are
“applied chapters” where the main extraction procedures used, as well as the appli-
cation of HRMS in pesticide residue analysis in several types of matrices, are
described. Finally, Chapters 8e10 describe new advances on the use of HRMS
such as gas chromatography (GC)eHRMS, ambient ionization techniques, and
identification of transformation products. The content of each chapter is described
in more detail as follows.
xiii
xiv Preface
To facilitate the introduction to the topics presented in this book, Chapter 1 de-
scribes the principles of HRMS, explaining several concepts such as monoisotopic
mass, resolution, mass accuracy, isotopic pattern, etc. Moreover, the differences be-
tween low- and high-resolution mass spectrometry are also discussed. In Chapter 2,
the several analyzers that could be used in HRMS are described, indicating the dif-
ferences between the HRMS systems from different vendors, as well as the software
that, nowadays, is available to process all the data provided by these analyzers.
Finally, online resources such as ChemSpider and MassBank are described. Target,
nontarget, and unknown analysis strategies are explained in Chapter 3, describing
how a database is prepared and the workflow commonly used for nontarget and un-
known analysis. Chapter 4 provides a complete overview of the general require-
ments indicated by international guidelines (i.e., SANTE) regarding the use of
HRMS in pesticide residue analysis, as well as the parameters that should be vali-
dated. A section describing pesticides legislation (indicating MRLs) is also included.
Bearing in mind that theoretically, unlimited number of compounds could be deter-
mined by HRMS, Chapter 5 describes the development of generic extraction
methods that allow the simultaneous extraction of a huge number of pesticides
that are needed. Chapters 6 and 7 cover the main applications describing the use
of HRMS coupled with UHPLC during the analysis of pesticide residue analysis
in fruits and vegetables (Chapter 6) and in food from animal origin (Chapter 7).
Although most of the current applications focused on pesticide residue applying
HRMS use liquid chromatography as the separation technique, in the last few years,
GC has also been utilized, because of the development of new ionization sources,
such as atmospheric pressure gas chromatography or new couplings such as GC-
Orbitrap. Therefore, Chapter 8 is devoted to this topic to highlight the potentiality
of GCeHRMS in pesticide residue analysis as well as new approaches such as
ion mobility. Because of the potentiality of HRMS, chromatographic step could
be removed from the conventional analytical method, and rapid detection of the
target compounds could be performed. This approach is interesting for pesticides
that cannot be commonly analyzed by multiresidue methods (i.e., very polar pesti-
cides), and the use of HRMS could simplify the analytical strategy. Chapter 9 is
focused on this approach as well as in the use of ambient mass spectrometry, which
allows for the direct analysis of samples without sample extraction and chromato-
graphic separation, especially when HRMS analyzers are used. Finally, Chapter
10 is dedicated to the advantages that HRMS provides for the identification of pesti-
cide transformation products.
The book is intended for researchers and professionals working with LCeMS,
such as food chemists, analytical chemists, toxicologists, food scientists, and
everyone who uses/needs this technique to evaluate food safety. Moreover, under-
graduate students would also be interested.
Finally, it has been a great pleasure to thank all the authors of this book for their
work. All of them are specialists and we really appreciate their effort and time. We
also give special thanks to the editorial and production teams of the publisher,
Preface xv
Elsevier, especially Karen R. Miller, who started this adventure, and Jackie Trues-
dell, for her patience and help, allowing this work to come to fruition.
Roberto Romero-González
Antonia Garrido Frenich
Almerı´a, Spain
November, 2016.
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CHAPTER
• Atomic mass: The number that represents the element’s mass based on the
weighted average of the masses of its naturally occurring stable isotopes. For
example, the integer atomic mass of bromine is 80 Da. This is because there are
only two naturally occurring stable isotopes of bromine, 79Br and 81Br, which
exist in nature in about equal amounts. When the relative mass (Mr) of an ion,
molecule, or radical is reported, it is based on the atomic masses of its elements.
• Nominal mass: Mass of a molecular ion or molecule calculated using the isotope
mass of the most abundant constituent element isotope of each element
(Table 1.1) rounded to the nearest integer value and multiplied by the number
of atoms of each element. Example: nominal mass of H2O ¼ (2 1 þ 1 16)
u ¼ 18 u.
• Monoisotopic mass: Exact mass of an ion or molecule calculated using the mass
of the most abundant isotope of each element. Example: monoisotopic mass of
H2O ¼ (2 1.007825 þ 1 15.994915) u ¼ 18.010565 u. The exact mass of
the common elements and their isotopes are provided in Table 1.1.
• Exact mass: Calculated mass of an ion or molecule with specified isotopic
composition.
• Mass defect: Difference between the nominal mass and the monoisotopic mass of
an atom, molecule, or ion. It can be a positive or negative value.
• Relative isotopic mass defect (RDm): It is the mass defect between the mono-
isotopic mass of an element and the mass of its Aþ1 or its Aþ2 isotopic cluster
(Thurman & Ferrer, 2010). For instance, RDm for the pair 35Cl:37Cl is
0.0030 Da.
• Average mass: Mass of an ion or molecule weighted for its isotopic composition,
i.e., the average of the isotopic masses of each element, weighted for isotopic
abundance (Table 1.1). Example: average mass of
H2O ¼ (2 1.00794 þ 1 15.9994) u ¼ 18.01528 u.
• Accurate mass: Experimentally determined mass of an ion of known charge.
• Mass accuracy: Difference between the mass measured by the mass analyzer and
theoretical value.
• Resolution or mass resolving power: Measure of the ability of a mass analyzer to
distinguish two signals of slightly different m/z ratios.
• Mass calibration: Means of determining m/z values of ions from experimentally
detected signals using a theoretical or empirical relational equation. In general,
this is accomplished using a computer-based data system and a calibration file
obtained from a mass spectrum of a compound that produces ions of known m/z
values.
• Mass limit: Value of m/z above or below which ions cannot be detected in a mass
spectrometer.
• Mass number: The sum of the protons and neutrons in an atom, molecule, or ion.
If the mass is expressed in u, mass number is similar to nominal mass.
• Most abundant ion mass: The mass that corresponds to the most abundant peak
in the isotopic cluster of the ion of a given empirical formula.
1.1 Introduction (To High-Resolution Mass Spectrometry) 3
Table 1.1 Nominal, Isotopic, and Average Masses of Some Common Stable
Isotopes
Nominal Isotopic Average
Element Isotope Abundance Mass Mass Mass
1
H H 99.9885 1 1.007825 1.00794
2
H 0.0115 2 2.014102
12
C C 98.93 12 12.000000 12.0110
13
C 1.08 13 13.003355
14
N N 99.632 14 14.003074 14.00674
15
N 0.368 15 15.000109
16
O O 99.757 16 15.994915 15.9994
17
O 0.038 17 16.999131
18
O 0.205 18 17.999160
19
F F 100 19 18.998403 18.9984
23
Na Na 100 23 22.989770 22.9898
28
Si Si 92.2297 28 27.976927 28.0855
29
Si 4.6832 29 28.976495
30
Si 3.0872 30 29.973770
31
P P 100 31 30.973762 30.9738
32
S S 94.93 32 31.972072 32.0660
33
S 0.76 33 32.971459
34
S 4.29 34 33.967868
35
Cl Cl 75.78 35 34.968853 35.4527
37
Cl 24.22 37 36.965903
79
Br Br 50.69 79 78.918336 79.9094
81
Br 49.32 81 80.916289
127
I I 100 127 126.904476 126.9045
5
6 CHAPTER 1 HRMS: Fundamentals and Basic Concepts
FIGURE 1.1
Methods of calculating mass resolving power.
Reprinted from Picó, Y. (2015). Advanced mass spectrometry. In Y. Picó (Ed.), Comprehensive analytical
chemistry, Vol. 68. Amsterdam: Elsevier, with permission from Elsevier.
FIGURE 1.2
Fragmentation pattern for the flonicamid pesticide (TFNA: 4-trifluoromethylnicotinic acid;
TFNA-AM: 4-trifluoromethilnicotinamide).
1.4 Mass Calibration in High-Resolution Mass Spectrometry 9
FIGURE 1.3
Internal mass calibration and target mass detection during a multiple ion detection (MID)
process in a double focusing system (DFS) instrument: A, magnet locking and lock mass
sweep. Mass calibration and resolution determination; B, electrical jump to calibration
mass; C, calibration mass sweep and mass calibration; D, electrical jumps to target
masses; E, electrical jump to calibration mass for mass calibration.
12 CHAPTER 1 HRMS: Fundamentals and Basic Concepts
wer of at least 5500 is needed to separate C13 H16 eC2 H4 þ and C13 H16 eCOþ ions,
even if the difference between both masses is 0.036385 u. Another difficulty is that
the possible elemental composition becomes quite larger as the mass increases.
Full scan can be one of the simplest acquisition modes to obtain an accurate mass
value when an internal mass calibration is carried out. Accurate masses of all ions
can be determined in one single chromatographic run, and elemental composition
can be achieved with an adequate accuracy of less than 5 ppm. Peak-matching
mode is a more accurate mass measurement technique with a typical accuracy lower
than 0.3 ppm. For that, only one ion is determined at a time, for example, using the
MID technique described earlier. It is recommended that the mass of the mass cali-
bration ion must be within 2% of the unknown mass to give the highest accuracy. A
combination of slow scanning of the accelerating voltage and computer programs
that improve signal averaging, smoothing, and peak centroiding improves mass ac-
curacy (Hammar, Pettersson, & Carpenter, 1974).
A nominal mass may result from several combinations of elements, but only one
composition can match an accurate mass. Nevertheless, more possible combinations
can occur with an increased number of atoms in a molecule. A list of potential mo-
lecular formulas for various masses has been proposed (Beynon & Williams, 1963).
Diverse algorithms and computer programs are available for elemental composition;
many of them are available online.
Finally, it should be mentioned that RDm is also a valuable tool for confirmation
purposes. The sum of RDm of all isotopes of the molecule is defined as the isotopic
mass average (IMA), and Padilla-Sánchez et al. (2012) used this approach for the
reliable identification of small pesticides such as ethephon and N-acetyl-glufosinate,
obtaining an experimental IMA(Aþ2) value of 0.00286 Da for ethephon, which was
within the theoretical interval of 0.0029 0.0001 Da.
References 13
ACKNOWLEDGMENTS
The authors gratefully acknowledge Andalusian Regional Government (Regional Ministry of
Innovation, Science, and Enterprise) and FEDER for financial support (Project Ref. P-12-
FQM 1838).
REFERENCES
Beynon, J. H., & Williams, A. E. (1963). Mass and abundance tables for use in mass
spectrometry. Amsterdam: Elsevier.
Bristow, A. W. T., & Webb, K. S. (2003). Intercomparison study on accurate mass measure-
ment of small molecules in mass spectrometry. Journal of the American Society for Mass
Spectrometry, 14(10), 1086e1098.
Busch, K. L. (2004). Masses in mass spectrometry: Balancing the analytical scales.
Spectroscopy, 19(11), 32e34.
Busch, K. L. (2005). Masses in mass spectrometry: Perfluors and more. Part II. Spectroscopy,
20(2), 76e81.
Chernushevich, I. V., Loboda, A. V., & Thomson, B. A. (2001). An introduction to
quadrupole-time-of-flight mass spectrometry. Journal of Mass Spectrometry, 36(8),
849e865.
Dass, C. (2007). Fundamentals of contemporary mass spectrometry. Hoboken, New Jersey:
John Wiley & Sons, Inc.
Dienes, T., Salvador, J. P., Schürch, S., Scott, J. R., Yao, J., Cui, S., et al. (1996). Fourier trans-
form mass spectrometrydadvancing years (1992-mid. 1996). Mass Spectrometry Re-
views, 15(3), 163e211.
Ekman, R., Silberring, J., Westman-Brinkmalm, A., & Kraj, A. (Eds.). (2009). Mass spectrom-
etry. Instrumentation, interpretation, and applications. Hoboken, New Jersey: John Wiley
& Sons, Inc.
Ferrer, I., & Thurman, E. M. (2009). Liquid chromatography time-of-flight mass spectrometry e
principles, tools, and applications for accurate mass analysis. Hoboken: Wiley-
Interscience.
Gross, J. H. (2011). Mass spectrometry. A textbook (2nd ed.). Berlin Heidelberg: Springer.
Gross, M. L. (1994). Accurate masses for structure confirmation. Journal of the American So-
ciety for Mass Spectrometry, 5(2), 57.
Hammar, C. G., Pettersson, G., & Carpenter, P. T. (1974). Computerized mass fragmentogra-
phy and peak matching. Biomedical Mass Spectrometry, (1), 397e411.
Herbert, C. G., & Johstone, R. A. W. (2003). Mass spectrometry basics. Boca Ratón: CRC
Press.
de Hoffmann, E., & Stroobant, V. (2007). Mass spectrometry. Principles and applications (3rd
ed.). Chichester: John Wiley & Sons Ltd.
IUPAC. Compendium of chemical Terminology, 2nd ed. (the “Gold Book”). Compiled by A.
D. McNaught and A. Wilkinson. Blackwell Scientific Publications, Oxford (1997). XML
on-line corrected version: http://goldbook.iupac.org (2006) created by M. Nic, J. Jirat, B.
Kosata; updates compiled by A. Jenkins.
Kim, S., Rodgers, R. P., & Marshall, A. G. (2006). Truly “exact” mass: elemental composition
can be determined uniquely from molecular mass measurement at w0.1 mDa accuracy
14 CHAPTER 1 HRMS: Fundamentals and Basic Concepts
2.1 INTRODUCTION
Undoubtedly, the establishment of electrospray ionization (ESI) in the late 1980s (Fenn,
Mann, Meng, Wong, & Whitehouse, 1989, p. 64; Yamashita & Fenn, 1984, p. 4451)
and its enormous potential for biomedical applications prompted the development and
growth of liquid chromatographyemass spectrometry (LCeMS) instrumentation.
Several manufacturers launched LCeMS instrumentation in the mid-1990s with
either ion trap or triple quadrupole (QqQ) analyzers, which established itself as the
gold standard for quantitative trace analysis. Meanwhile, the use of high-resolution
LCeMS instrumentation with time-of-flight (TOF) analyzers was commercially
available from 1996 with the development of first hybrid quadrupole time-of-flight
(Q-TOF) instruments. Micromass commercialized the first Q-TOF mass spectrometer
in 1996 (Q-TOF-1), which was furnished with ESI source, quadrupole mass filter,
hexapole collision cell, orthogonal acceleration, and microchannel plate detector. It
featured a resolving power of 5000. Unfortunately, these instruments were originally
applicable only to qualitative purposes such as the determination of molecular mass
and elemental composition through accurate mass analysis. There were several issues
(linear dynamic range due to detector saturation and scarce robustness) that limited
the use of such instrumentation for quantitative purposes.
The last two decades have witnessed an unprecedented growth of mass
spectrometric instruments of increasing high-mass resolving power. Such ability
enabling accurate mass measurements permits the determination of elemental
compositions for small molecules up to 500e1000 Da in mixtures containing
thousands of chemical components (Marshall & Hendrickson, 2008, p. 579). There
has also been an outstanding growth in the use of high-resolution instrumentation for
quantitative application. This fact has been supported by relevant improvements of
instrument performance in terms of resolution, sensitivity, and speed. Among them,
in 2004, several vendors (Agilent, Bruker, Waters) offered updated and upgraded
versions of TOF instruments, with improvements on electronics to prevent detector
saturation, temperature control of flight tube, and continuous accurate mass
calibration systems (dual sprayer, MassLock, etc.), which broadened the range of
applications and enable more versatile and user-friendly operation. In 2005, the
2.2.1 TIME-OF-FLIGHT
TOF is a relatively mature technology (Fjeldsted, 2016, p. 19). Actually, the concept
of TOF mass analysis was first proposed by Stephens in 1946 (Stephens, 1946, p.
691), and subsequently developed by Cameron and Eggers and referred to as a
“velocitron” in 1948 (Cameron & Eggers, 1948, p. 605). The basic principle of
TOF instrument relies on the velocity-dependent separation and subsequent detection
of ions as they travel through a flight tube. Charged ions produced are accelerated into
TOF tube and are allowed to drift along the path. Provided that velocity is dependent
on mass-to-charge (m/z) ratio, the lighter ions will fly faster and reach the detector
earlier. There are two main types of TOF analyzers: linear and of orthogonal
acceleration. Whereas the use of linear time-TOFs was associated with pulsed
ionization methods such as laser beams with matrix-assisted laser desorption/
ionization (MALDI) experiments, the latter is the most commonly used configuration
nowadays. Fig. 2.1 shows the scheme of an orthogonal acceleration TOF. It consists of
FIGURE 2.1
Scheme of an orthogonal acceleration time-of-flight instrument.
18 CHAPTER 2 HRMS: Hardware and Software
an ionization interface, which is followed by an ion transfer section (ion optics) and
focusing region. The continuous ion beam emerging from the ion source is sharpened
within the ion optic path and sent toward the pulser. A high-voltage pulse is applied to
the pusher plate to orthogonally accelerate pockets of ions into the flight tube.
Wiley and McLaren (1955, p. 1150) realized the importance of space focusing.
They found that small differences on the position of the ions created in the ion source
and the distances to the detector affected resolving power dramatically. Ions created
at the back of the ion source volume had a longer distance to travel to the detector
than those that were created closer to the front of the ion source volume. They
demonstrated that with the appropriate increase in voltage at the back pusher plate
within the ion source it was possible to significantly reduce the loss of resolution
because of the origin of the ions within the source. This advance was connected
to the early development of the first TOF commercial instrument offered by Bendix
in the mid-1950s (Fjeldsted, 2016, p. 19; Wiley, 1956, p. 817).
Therefore, during TOF analysis, it is important that all ions receive an equal
acceleration to, as much as possible, reduce spreading in time. The focusing of
the ion beam before reaching the pusher and the following orthogonal acceleration
become critical to ensure equal starting conditions for all ions regardless of their
individual masses and thermal energy. Thus, the initial spatial and ion energy spread
among the ions are minimized. Mamyrin (1966) proposed the use of an ion mirror
(referred to as a reflectron) in the flight path to compensate for the spread in the
initial energy of the ions. Mamyrin showed that when a proper reflecting field
was established, ions with greater energy proportionally travel further into the
reflectron before being reflected with the effect that the arrival time of the ions at
the detector shows a greater tolerance to the effects of their initial energy spread.
Therefore, a careful design of the reflectron permits a compensation of speed
differences, as caused by the remaining energy difference among the accelerated
ions. In addition, the implementation of the reflectron doubled the flight path length
to achieve longer flight times and therefore increased resolution.
TOF instruments are of pulsed nature. The use of ion packets is central to TOF
analyzers. Initially, the creation of ion packets was generated by employing a pulsed
ionization source such as an electron ionization filament or a beam deflector. In the
latter approach, a pulsed ion packet was created through deflection by gating, or
momentarily steering, the ion beam into the TOF analyzer. In this sense, the concept
of orthogonal acceleration was proposed by Dawson and Guilhaus (1989, p. 155). A
continuous beam of ions was momentarily pushed in a direction perpendicular to the
direction of the continuous ion beam. The packet of orthogonally directed ions
enters the TOF tube that is therefore oriented perpendicular to the continuous ion
beam rather than in-line. With this configuration, the energy spread is substantially
minimized. The introduction of the orthogonal TOF geometry was of paramount
importance for adapting TOF to atmospheric pressure ionization sources, such as
ESI, and the subsequent growth in TOF applications in LC/MS.
The detectordlocated at the end of the flight pathdrecords the arrival time and
the number of incoming ions. The square of the flight time is proportional to the m/z
2.2 Principles of High-Resolution Mass Spectrometry Analyzers 19
of the detected ion. Hence, masses (m/z) can simply be calculated after an instrument
calibration process. Because of the fast ion flight times, a single push cannot be used
to record a spectrum. Hundreds of consecutive pushes are summed to produce a
single, averaged spectrum instead (corresponding to one chromatographic data
point). A single pulser push accelerates only the ions passing above the surface of
the pusher plate. The next push should not be started before the heaviest ion
accelerated by the preceding push has reached the detector. This results in duty
cycles clearly below 100%. The very short ion flight times within the flight tube
put extreme importance on the frequency of the detection device and the following
signal processing. In the past, fast time-to-digital conversion (TDC) detectors were
only able to measure the flight time, but could not count the number of the
simultaneously incoming ions at a given time. Such devices have been replaced
by analog-to-digital conversion (ADC) detectors, which unlike TDC devices can ac-
count for the number of ions simultaneously hitting the detector plate, thus enabling
the expansion of the dynamic range to up to four decades. Yet this point remains as
one of the main weaknesses of TOF detectors; they undergo saturation from a
threshold intensity, which not only affects the measured signal amplitude but also
can result in a relevant mass shift of the ions being affected by saturation.
the rotation of the ions at a cyclotron frequency [nc (Hz)] in the spatially uniform
magnetic field (B) is:
nc ¼ ezB=2pm
in which B is the magnetic field, m is the mass, and e is the elementary charge. At
room temperature, typical ion cyclotron orbital radii are at the submillimeter level.
Considering that ions with a certain m/z rotate with random phase, it is necessary to
resonantly excite the ions with an oscillating or rotating electric field to yield a
spatially coherent packet of ions of each given m/z. The motion of the different
ion packets gives rise to a time-domain signal consisting of the difference in current
induced on a pair of opposed electrodes (Fig. 2.2).
Nowadays, only one vendor commercializes a portfolio of FT-ICR instruments
(Bruker Daltonics, Bremen, Germany). Up to three different models are offered
including a hybrid quadrupole FT-ICR 7-Tesla (T) (solariX XR) and 12-T or 15-T
high-field solariX XR with resolving power up to 600,000 at m/z 400 with 1 s
transient (and up to 10,000,000 resolving power depending on magnet and
acquisition time). To enable a flexible range of experiments, these instruments
permit a wide range of fragmentation techniques including in-source collision-
induced dissociation (CID), CID in collision cell, electron-capture dissociation
(ECD), electron-transfer dissociation (ETD), and sustained off-resonance irradiation
(SORI) CID in cell. In the past, Thermo also offered a hybrid linear ion trap (LTQ)-
FT-ICRMS, but it was withdrawn in 2014, with the emergence of high-field
Orbitrap, which nearly matched the performance of FT-ICR systems. The
applications foreseen for this type of instrumentation are petroleomics (Cho,
Ahmed, Islam, & Kim, 2015, p. 248; Nikolaev, 2015, p. 421; Schwemer, Rüger,
Sklorz, & Zimmermann, 2015, p. 11,957) and state-of-the art proteomics research
(Marshall & Chen, 2015, p. 410; Nicolardi, Bogdnov, Deelder, Palmblad, & Van
FT
0
Der Burgt, 2015, p. 27,133), being the application of FT-ICR in pesticide testing
scarcely addressed as the requirements and maintenance cost of such sophisticated
instrumentation are not met by standard routine laboratories (Gilbert-López et al.,
2013, p. 419).
2.2.3 ORBITRAP
The Orbitrap mass analyzer is the first high-performance mass analyzer that employs
trapping of ions in electrostatic fields, together with a sophisticated ion injection
process, which enables high resolution, mass accuracy, and excellent sensitivity
for addressing numerous analytical applications in both research and routine
analysis. The principle of Orbitrap is based on the Kingdon ion trap described in
1923 (Kingdon, 1923, p. 408). It was a trapping device consisting of a charged
wire stretched along the axis of an enclosed metal can. The wire establishes an
electrostatic field within the can, and ions that possess sufficiently high tangential
velocity orbit the wire, rather than directly colliding with it (Eliuk & Makarov,
2015, p. 61). It was first commercially introduced in 2005 (Eliuk & Makarov,
2015, p. 61). It provides a very convenient balance between the advantages of a
benchtop instrument featuring high resolution approaching FT-ICR performance,
whereas the instrument size, laboratory space requirements, and regular operational
maintenance are more comparable to Q-TOF instruments.
A thorough explanation of the mass analysis process is detailed elsewhere (Eliuk
& Makarov, 2015, p. 61). This mass analyzer is based on the confinement of ions in
an electrostatic potential well (Makarov, 1999, US 5886346; Makarov, 2000, p.
1156; Makarov & Hardman, 2006, US 6998609 B2) created between two carefully
shaped electrodes: an inner coaxial spindled-shaped electrode and an outer (barrel-
like) electrode, which is actually composed of two symmetrical halves electrically
isolated from each other, set out for two purposes: establishment of the ion trapping
fields and as receiver plates for image-current detection. The electrodes are precisely
machined so that the electrostatic attractions of the ions to inner electrode are finely
balanced by centrifugal forces, which cause the ions to orbit around the spindle.
Previously, ions are first accumulated on an external injecting device (C-trap) that
traps the ions in gas-filled quadrupole being then injected tangentially into the
mass analyzer in short pulses (Fig. 2.3). Besides, an axial field causes the ions to
oscillate harmonically along the spindled-shaped electrode. The outer electrodes
allow differential image-current detection. These image currents produced by the
oscillating ions are detected, followed by a fast Fourier transform (FT) to convert
the time-domain signal to frequency domain and then to m/z spectrum. Here the
resolution is directly proportional to number of harmonic oscillations detected.
The resolving power can be enhanced by increasing the gap between inner and outer
electrodes providing higher field strength for a given voltage. As the maximum
acquisition time is limited in Orbitrap, the resolution power is not as high as
FT-ICR. However, commercial Orbitrap analyzer provides a nominal resolution
power as high as 500,000 at FWHM (Table 2.1).
22 CHAPTER 2 HRMS: Hardware and Software
FIGURE 2.3
Cross section of the C-trap ion accumulation device and the Orbitrap mass analyzer with
an example of an ion trajectory. During the voltage ramp, the ion packets enter the
Orbitrap mass analyzer forming rings that induce current which is detected by the
amplifier.
Reprinted with permission from Thermo Fisher Scientific, Copyright © 2016.
23
24 CHAPTER 2 HRMS: Hardware and Software
FIGURE 2.4
Key elements of modern Quadrupole time-of-flight (Q-TOF) mass spectrometer.
Reprinted with permission from Elsevier, Copyright © 2016 [Fjeldsted, J.C. (2016). Advances in time-of-flight
mass spectrometry. In S. Pérez, P. Eichhorn, & D. Barceló (Eds.), Comprehensive analytical chemistry. Appli-
cations of time-of-flight and Orbitrap mass spectrometry in environmental, food, doping and forensic analysis
(p. 19). Amsterdam: Elsevier].
The use of radio frequency (RF) fields exerts a force on ions over a wide range of
pressure and mass. Based on this principle, the QJet ion guide designed by Sciex
improved efficiency in separating ions from the noncharged species (neutrals),
improving the focusing of captured ions into the mass spectrometer. Another
approach (StepWave) was presented by Waters and first implemented in 2008 in
the Synapt G2 hybrid instrument based on the use of a set of ion guides to shift
the ion beam axis offset to the atmospheric pressure sampling inlet. RF and direct
current (DC) potential are applied onto a set of ion concentric rings with overlapping
apertures, so that the ions are shifted off the main axis into smaller off-axis
concentric apertures, which focus the ion beam and direct it to the subsequent
mass analysis stages while neutral gas passes along the principle axis being diverted
to the exhaust.
Based also on the use of stacked ring RF ion guides, R.D. Smith et al. showed
that ion confinement could be achieved at pressures in the range from 0.1 to
30 torr (Kelly, Tolmachev, Page, Tang, & Smith, 2010, p. 294), when constructing
26 CHAPTER 2 HRMS: Hardware and Software
a set of ring electrodes having a linear decrease in the inner diameters in the last sec-
tion of the stack (the so-called ion electrodynamic funnel), which could be used indi-
vidually or as a pair in succession (dual ion funnel) with the second having a lower
operating pressure than the preceding funnel. These RF ion funnel technologies
enable highly efficient ion capture and may provide up to a 10-fold increase in
the atmospheric sampling efficiencies and overall sensitivity (Fjeldsted, 2016, p.
19). The use of multiplexed (hexabore) capillaries has also been proposed to in-
crease ion sampling efficiency (Kelly et al., 2010, p. 294).
Resolving power and flight path length. Unlike Orbitrap, the resolving power
in TOF analyzer increases with m/z values. Several solutions have improved the
overall performance of commercial TOF instrumentation particularly in terms of
resolving power, attaining values up to 50,000 or even higher. Most of the improve-
ments should be attributed to both ion optics (focusing of ions before TOF analysis)
and detection electronics including the increase in the detector operation frequency
of ADC TOF detectors (from 1 to 5 GHz) (Fjeldsted, 2016, p. 19). Besides, there is
another obvious strategy that increases resolution (increasing the flight path length),
although at the expense of sensitivity. This can be accomplished by using either
longer flight tubes (up to 1.5 m) or multiple reflection by placing a second mirror
between the ion pusher and detector, reflecting the beam twice, thus doubling the
effective flight path length [such as W optics mode in Waters, N-optic design (Sciex)
or folded path technology enabling up to 64 reflections (LECO)]. Ion mirror
(reflectron) has also been improved through the use of two-stage mirrors, which
compensate ion energy spread from the ion pusher. Nevertheless, reflectrons not
only double the flight path length, but also are responsible for a relevant loss of
recorded ion abundances. Hence, users of such multireflectron instruments have
to come across a compromise between sensitivity and selectivity. Last, but not least,
flight tube temperature control in the system also reduces the internal energy spread
of the ions with the same mass, so that an increase in resolution is also achieved.
Ion detection. Ion detection and digitization have also undergone significant
improvements in the last decade. It seems that most of the manufacturers have found
that multichannel plateebased detectors with ADC are the best choice to avoid
detector saturation and lack of linearity (Fjeldsted, 2016, p. 19). However, the use
of very fast TDC conversion and a multianode detector is also used in instruments
offering excellent performance such as Sciex TripleTOF 5600 Q-TOF (Fig. 2.5)
(Andrews, Simons, Young, Hawkridge, & Muddiman, 2011, p. 5442). A detailed
discussion on this matter is described by Fjeldsted (2003, 2016, p. 19).
Accurate mass measurements and mass calibration. Mass accuracy is central
to HRMS. This fact is particularly important in TOF instruments, as they are
generally less robust than FT-MS instruments in terms of the stability of accurate
mass measurements and the subsequent accuracy (ppm mass errors). This is a key
parameter, as small changes in the experimental conditions (such as laboratory
temperature) usually affect the actual time measurements of ions TOF, provided
the change in drift tube path length due to temperature changes (small thermal
expansion or contraction of the flight tube length). A high-performance temperature
2.3 Time-of-Flight Mass Spectrometry 27
FIGURE 2.5
(A) Schematic of a Quadrupole time-of-flight instrument from Sciex (TripleTOF 5500)
using multianode time-to-digital conversion (TDC) detection. (B) An image of the
machined TripleTOF MS instrument platform.
Reproduced with permission from American Chemical Society, Copyright © 2011.
control assembly of the drift tube is mandatory to assess mass accuracy performance
of TOF instruments.
For this purpose, mass axis calibration is an important process for obtaining
reliable results. Mass calibration consists of comparing the observed experimental
mass measurement of one or two known ions with their (theoretical) exact mass.
Calibration may be internal (i.e., the reference masses are for ions of known
elemental composition in the same mass spectrum of the analyte) or external (i.e.,
reference masses from a mass spectrum of another analyte acquired under similar
conditions). Internal calibration is typically at least twofold more accurate than
external (Marshall et al., 1998, p. 1). Accurate mass correction is usually performed
in full-scan (FS) data, as the ions selected are available in all acquired spectra
through the run. However, when recording accurate mass spectra in MS/MS mode
with precursor ion selection, the lack of such calibrating ions hampers the accuracy
of product ion spectra.
In the first generation of TOF instruments, external mass calibration was applied
before the chromatographic run. This approach did not deliver results better than
5e10 ppm relative mass error because of the significant drifting of instrumental
status (e.g., flight tube temperature). To overcome this situation, various strategies
have been proposed to provide continuous accurate mass calibration in commercial
28 CHAPTER 2 HRMS: Hardware and Software
TOF instruments, allowing them to feature routine relative mass errors in the range
of 1e3 ppm, when using mass calibration devices such as MassLock (Waters), the
dual sprayer solution (Agilent Technologies), or similar technologies presented by
other manufacturers such as the TwinSprayer (Sciex), which is an independent
calibrant delivery path proposed in the recently commercialized compact Sciex Q-
TOF (Sciex X500R Q-TOF) dedicated for small molecule applications (Fig. 2.6).
These assemblies provide a compensating mechanism to keep flight times constant
for a given fixed mass (reference solution). First, Waters proposed the discontinuous
periodical infusion of a lock mass solution by a switching device that alternatively
permitted the flow from two independent ESI sprayers (the reference solution and
the analytical sample) (Eckers et al., 2000, p. 3683; Wolff et al., 2001, p. 2605).
This strategy is not flawless because it introduces mass signalsdunrelated to the
sampledinto both the spectrum and the data file, which are used to calibrate the
entire data set at the end of the run. Likewise, Agilent proposed the use of a dual
sprayer assembly, where a calibrant solution was continuously sprayeddorthogonal
to the analytical sprayerdand introduced in the mass spectrometer (Fig. 2.6). This
solution provided excellent results even enabling the measurement of the mass of an
ion using twin fragment ions (Ferrer & Thurman, 2005, p. 3394). However, although
they are orthogonal, there might be some interaction between the analytical and
reference sprayers, which may yield signal suppression if the concentration/
abundance of the reference solution analytes is not controlled. As mentioned
previously, the newest Sciex Q-TOF (Sciex X500R Q-TOF) makes use of an
FIGURE 2.6
Examples of continuous calibration systems for mass calibration in time-of-flight
instrumentation: (A) Agilent MSD TOF (2004); (B) Sciex X500R (2016).
Reprinted with permission from Sciex and Agilent Technologies, Copyright © 2016.
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pioneers. They splashed out of the water on to the sands, and
started towards the city at a run. As they ran, they heard Pain
keeping back the ranks till the charge should have been fired. The
feet splashed behind them slowly, growing fainter. The fire on the
beach grew brighter, they were passing it; the walls were before
them, only a hundred yards away. “Against the door,” he said,
panting. “Against the hinges. One keg spilled below.” The town was
aroused now. They could hear the cries and hurry. Still no sign came
from the walls. “Sentinel’s asleep,” said one of the men. “There,” said
another. “Up with her. Up with her. We’re seen.” A man showed upon
the wall by the gate. “Ahi,” he screamed. “Piratas. Piratas. Piratas.
Cuidado. Cuidado.” He fired his gun into the air; the flash shot up like
the flash of a blast-charge. There came cries and a noise of running.
A few heads showed. The wall spouted fire in a volley. They were up
against the wall, against the iron-plated door, piling the kegs against
the hinges, tamping them down with sods and stones. Margaret
snatched one keg and spilled it along the door-sill. “There,” he said.
“There. Now your fuse, fuseman.” The quick-match was thrust into a
keg. “Up along the walls, boys. Quick. Scatter. Pronto.” He thrust
them sideways. They saw what he wanted. When the imaginations
are alert there is little need for speech. No man could have heard
him. The racket in the town was uproar like earthquake. The whole
wall above them was lit with fire spurts. Mud and plaster were
tinkling in a rain upon them. They ran fifty yards like hares, paying
out the quick-match. “Now,” said Margaret. The match flashed. A
snake of fire rippled from them. They saw the shards of pots gleam,
then vanish. They saw old bones, old kettles, all the refuse below the
walls. “Down,” he shouted. “Down.” They flung themselves down.
The beach to their left flashed, as the pirates fired at the wall.
There came a roar, a rush of fire, a shaking of the land. Mud, brick,
stone, shards of iron and wood, all the ruin of the gate, crashed
among them, flying far among the trees, thumping them on their
backs as they lay. After the roar there was a dismayed silence. A
wail of a hurt man sounded, as though the wrecked gate cried. Then
with a volley the privateers stormed in. From where he lay, Margaret
could watch them plainly; the dawn had broken. He saw them
charging, tripping in the sand, their gun-barrels glinting. An Indian led
them, a screaming Indian, who danced and spun round, waving his
machete. “Lie still,” he shouted to his pioneers. “Let them pass.
They’ll shoot you down. Lie still.” With yells and shots the storming
party swept up the ruin. “Up with her,” they shouted. “Up with her.”
They were clambering over the wreck, tripping, stumbling, kneeling,
to fire, clubbing at the guard. “Now,” said Margaret, drawing his
sword. They rose up from their nest among the tip. They were with
their fellows, they were climbing the heaped stone, amid smoke and
oaths and fire-lit faces. Margaret was inside Tolu. The south wall was
won.
The land-breeze, very faint now, drifted the smoke slowly. He
could see little. He could see in glimpses the whitewashed houses,
the line of the south road, a man with his back to a wall, a woman
fallen, figures rushing. He could see enough to know that the enemy
were making no stand. He ran on up the road to the Plaza, one of a
mob. Windows opened above him. People fired from the windows.
Women were flinging pots, tiles, braziers. Some one had begun to
ring the alarm bell in the church. The broken clang sounded out
above the screams and the firing. Now it was all clear before him. He
was in the Plaza, shouting to his mob to form. There in front of him
the troops were mustering. They were running from their quarters,
half clad, in rags, just as they had started from sleep. Heavy fire was
rolling at the north wall. Troops were running thither. In the centre of
the Plaza, about a cotton tree, a score of Spaniards were forming in
line. A halberdier was dressing them. They stood firm, handling their
guns, hearkening to their capitan. Margaret saw them clearly, and
praised them in his heart for the flower of soldiery. They were of the
old Spanish foot, the finest troops in the world. Even as he looked he
saw them falling forward. He was among them. His sword jarred him
to the shoulder as it struck on a gun-barrel. He saw them about him.
They were breaking. They were gathering into clumps. They were
being swept into the mob of citizens flying to the east gate.
Half an hour later the newly risen sun showed him a captured
town. In the streets, in the Plaza, everywhere, lay the dead and
dying. They lay in heaps in some parts. All the clutter and wreck of
war, the clouts, the cast arms, the gear flung away by the fleeing, lay
littered in the sands. The walls were chipped and starred with bullet
marks. The stink of powder was everywhere. Women still screamed.
Wounded men wailed where they lay, with the pitiful whimpering cry,
like that of a beaten hound, which sickens all who hear it of the glory
of war. Firing was still going on; but the fight was over; the town was
in the hands of the privateers.
Margaret found himself at the east gate with Pain. About a dozen
of the crew of the Broken Heart stood by him, waiting for orders. He
took their names, and told them off to look out at the city gates, and
to spike the guns on the sea-wall.
“I’m going to the Governor’s house there, to look for my friend,” he
said. “Where’s my trumpeter? Sound the assembly, trumpet. Muster
your men, will you, Captain Pain?”
The assembly sounded. The men fell in, answering to their names.
The boat-guard with the doctors landed. The captains checked off
their lists, scanning the ranks closely whenever a man failed to
answer. The men were powder-blackened; some of them were
wounded, many were cut about the head. They sent the boat-guard
with the doctors to search the streets for the dead and hurt. Thirty-
three men were missing, all of them, save one, from the party which
had stormed the south wall.
“Strengthen the guards at the gates,” Margaret said. “Captain
Tucket, you take the north gate. I’ll see to the east. Captain Pain, will
you send a dozen to the south? Keep a sharp look out.”
He picked his own guard and sent them off to their duty. The other
gate-guards fell out unwillingly. Some of the privateers were eating
their breakfasts in the ranks.
“I’m going to the Governor’s house now,” he said to Pain. “Call me
at once if the Spaniards send a trumpet. No straggling. No looting,
mind.” As he turned towards the Governor’s house he heard the men
behind him snigger. He heard a voice ask Pain if this was to be the
new rule, now that Springer’s Key was full of college gents. Pain told
the man to take a severe turn.
Margaret drew his pistols as he came near the house; for though
most of the inhabitants had fled, a few poor men and slaves still
lounged in the streets, having nothing to lose. A single man, richly
dressed, might tempt these gangrels. He hailed a negro, who sat in
the sun in the Plaza, sucking a wound in his wrist.
“Ho muchacho,” he said in his schoolboy Spanish. “Donde esta la
casa del Gobernador?”
The negro waved his unhurt hand towards the house with a
gesture full of dignity. Then he continued to suck his wound, like a
dog licking a hurt paw.
“Gracias,” Margaret answered. “Pero el hombre Inglès. Donde
esta?”
Again the negro waved his hand towards the house, pausing in his
suction exactly like a dog. “He,” he said; then bent to suck again.
The house of the Governor fronted the Plaza. It was a big house,
with a patio. The lower story had no outer windows, no door.
Margaret had to climb the stone steps to the balcony, where a
chained monkey leaped up and down in the sun, between bites at a
plantain. The door leading to the inner part of the house lay open,
just as the fugitives had left it. A woman’s shawl was on the floor.
One runner had upset the monkey’s saucer of water. A chair had
been upset. As Margaret entered the house, with his pistols cocked,
he saw something beneath the chair, something bright, which he
took to be a snake. It was the scabbard of a sword, flung aside in a
soldier’s hurry. Margaret, pausing on the threshold to listen,
wondered if Stukeley had flung it there. He listened intently,
expecting to see Stukeley coming from the darkness of the corridor.
His mind was busy with the thought of Stukeley. What was he to say
to him? What was he to do to him? Suppose Stukeley came out
fighting? “I must bring him back,” he repeated. “I must bring him
back. He must be brought back.” A step sounded on the stairs
behind him. It was West, one of the Broken Heart’s seamen.
“Beg pardon, sir,” the man said, “may I come with you? There’s
maybe some of these Dons in the house.”
“Yes,” Margaret answered. “Listen.”
They listened in the doorway for a moment; but the house was
still, save for the chinking of the monkey’s chain.
“Gone, sir, I guess,” said West.
“Come on, then,” Margaret said. “Cock your pistols and come on.”
They passed through the littered hall into the left-hand corridor.
The jalousied shutters were shut on the patio side; but the doors of
some of the rooms were open, giving light to the passage. It was a
barely furnished house, hung with very old Spanish leather, ant-
eaten and mothed and mouldy, falling to pieces. In the first room, a
sleeping-room, the mosquito-nets had been torn from the cots, and
lay wrecked on the floor with a silver chocolate service. In another, a
chair stood against the wall, where a man had stood to snatch arms
from a trophy. In another sleeping-room they found the clothes of a
man and woman by the cot-sides just as they had been laid the night
before, when the couple retired. It was like being in the presence of
death to walk that house. It was as though they were looking on the
corpse of a house, on a house dug up from the sands, the life of it
gone and forgotten, only the pathetic husks left, that had once been
helps to men. They opened a shutter and looked out upon the patio.
A goat was tethered there, crying to be milked. They heard the
stamping of horses. One horse was scraping with his forefeet
against the floor of his stall. There was no sign of Stukeley there, no
trace of him, nothing to mark his presence.
“Now the other corridor,” Margaret said. They retraced their steps,
walking on tiptoe, listening intently. The first room in the other
corridor was a dining-room, furnished with heavy Spanish furniture of
the great period. A lute lay on the table, among wineglasses half full
of wine, a box of Peruvian suckets, a box of candied quinces, a dish
of avocat pears. Some one had been playing the lute, the night
before. The unknown player had fitted a new string. The broken
string lay among the litter where it had been thrown. Flies were black
among the suckets. The air smelt of the stale gums which had burnt
out before a crucifix on the wall. A shaft of sunlight came through a
broken shutter. The dust quivered in it. On the floor, in its road of
glory, a column of ants marched, stumbling over crumbs. There was
much silver in the room. Over the side-board was a Zurbaran, too full
of personality to be religious. Margaret looked at it, sighing, thinking
that only lesser artists could save their souls. There was no trace of
Stukeley here. “Let’s get out,” he said. “He’s not here.”
“There’s three more rooms,” said West. They entered the
Governor’s office.
It was a barer room than the others. It contained a table and a few
chairs. There were papers on the table; a locked account book, a list
of resident Indians, a list of citizens capable of bearing arms, a diary
in cipher. Under the table, in a coffer, were more account books,
Cammock’s portolano, and a copy of the same, traced from the
originals, now nearly finished.
“You see, West,” said Margaret. “You bear witness that I take
these two books?”
“Yes, sir,” said West. He walked over to a corner and picked
something up from a chair.
“Isn’t this Mr. Stukeley’s, sir?”
“Yes,” said Margaret, crossing over swiftly. “It’s his hat. And there’s
his sword-belt.”
They stood together, looking at the things, wondering how lately
the owner had flung them there, as he came in, hot, for the onzas.
They felt him to be very near them there. It was as though he were
coming sneering towards them, his fine teeth showing. His very
words came into Margaret’s mind, with their exact inflections. “Found
much? Eh?” Those were the words he would use.
“Hark,” said West suddenly. They listened.
“What did you think you heard?”
“There’s some one speaking in the next room, sir.”
“Listen.”
In the hush, they heard a sound like a sob, a low murmur of words;
then a rustling, chinking sound.
“Like some one praying,” West said.
“Careful now,” said Margaret. “Come on after me.” They crept from
the office on tiptoe, their pistols ready. In the corridor a board
creaked beneath them. They paused guiltily, straining their ears to
listen. They heard some one cross the room quietly. Then the door
was flung open, letting a glare of light into the corridor. A priest stood
before them, holding up a crucifix. Within was a bed. A woman knelt
by the bed. Some one lay on the bed, covered with a cloth. Margaret
raised his hand, and the priest stepped back, looking at their faces
curiously.
“Donde esta el caballero inglès?” Margaret asked. “El señor
Stukeley?”
A faint smile showed upon the priest’s mouth.
“Here,” he said in good English. He twitched back the bed-cloth
reverently, to show the body of Stukeley lying dead. The face was a
dull yellow, the mouth was inflamed. There was no need for further
words.
“Vomito,” said the priest.
“Yes,” Margaret said, uncovering. “Vomito.”
“This morning,” the priest said.
The woman rose from the bedside, as though to drive them away.
She was a black-eyed, hawk-nosed woman, of a crude and evil
beauty. She was dressed in red and brown, in an outlandish style.
She spoke in gasps, dreadful to hear; using broken English, laced
with oaths and Spanish words. “Damn,” she said. “Damn perros.
Cabrones de piratas.”
“De quién es?” Margaret stammered, meaning “Who are you?” He
had no gift of tongues.
“Mrs. Stukeley,” the priest said. “The widow. His wife.”
“Me Anna,” said the woman. “Me ’is wife.”
Margaret bowed; words seemed useless. He was only conscious
of the horror of it. He had not been prepared for this. He had sat at
meat so many times with this corpse. He had seen him so often, full
of life and health, going with a laugh to sin, in the pride of the flesh.
Now a little thing, the bite of a fly, no more, had brought him to a
death among strangers, in this low cot in the wilds, with his beauty
turned to horror, and his strength, if anything, a fiery chain upon his
soul. There he lay, under an Indian cotton, gone to his reward so
soon. Margaret had hated him. He shuddered now to think how he
had hated him. Looking at him as he lay there, in all the hideousness
of death, he felt the remorse which a death brings. He felt ashamed,
as though he had struck the corpse by hating him whom it had
covered. “It was my fault,” he said to himself. “It should have been
otherwise. One ought to live with this before one.” He saw where he
had acted hastily, where he had failed. He knew all that he might
have done. What moved him most was the thought that Olivia had
loved this man, had loved him tenderly, and that he, Olivia’s lover
had never known his character, had never guessed what it was in
him which was beautiful to her. Now he would never know. Standing
by the side of the corpse, he tasted all the bitterness of one who has
failed to apprehend another, and learns of his failure too late.
Stukeley was dead now, the old life was dead now. He would to God
that he were dead in Stukeley’s place, and that the old life might be
lived again. He would to God that this man’s passage to death had
been made pleasanter. He blamed himself. He was touched and
humbled almost to tears. “If I had only understood,” he thought, “you
would not have cast yourself away thus.” Now he had this to tell
Olivia.
“You were not what I thought, Stukeley,” he murmured aloud,
looking down at the face. “You were not what I thought. You won her
love. You were her chosen.
“My God,” he added to himself, “you won her by that very quality of
certainty which made you cast her aside.” He stood there trying to
create, in his moment of tenderness, fit words with which to tell her,
words which might comfort her, staying in her heart. The words
which came to him seemed blunt and cold. The only help that he
could give to her was to bury this part of her with all reverent and
noble rites.
“He was the Governor’s secretary,” said the priest. “He was not
here long. Not long in the country. The vomito takes the new-
comers.”
“Ah,” said Margaret, starting. “How well you talk English. Look
here. Come aside here. We must bury him at once?”
“Before the sun,” the priest said, with a shudder.
“Where is the burial ground?”
He raised his hand in benediction over the corpse, then led
Margaret out of the room. West followed them, reverent and awed,
speaking in a hushed voice. The priest led them by a back way to
the patio, thence by a postern to a side street, a good two hundred
yards from the house. The burial-ground was hedged with stone,
over which some creepers had grown. Little green lizards were
darting among the creepers. They glittered like cut glass. The gate of
the cemetery swung open on hinges of raw hide. When they entered,
some large rats scuttled to their burrows among the graves.
“Much sickness here,” said the priest. “It is not good to dig deep in
the ground.”
“I must dig this grave deep,” Margaret answered. “Look at the
rats.”
“They are large,” the priest said. “Much sickness in this poor town.”
“Where can I find a spade?” Margaret asked.
“Who knows?” said the priest. “You will tell your men to find one?
Ah?”
“I must do this myself,” he answered.
“But your men on the wall,” said the priest. “And your ship there.
Ah?”
Margaret looked towards the west, over the low sea-wall. Some of
his men were spiking the guns on the platform. He could hear the
click of the malls upon the spikes, as they snapped the soft iron flush
with the gun. Beyond them, very far away, were the ships; the tide
and the land-wind had set them out to sea again.
“They are waiting for the sea-breeze,” said the priest. “No getting
in till chocolate.”
“No,” said Margaret. “Now take me, please, to find a spade.”
“You are not a privateer?” the priest asked. “You do not sack us?”
“No,” he answered. “I am not a pirate. I demand the right to trade,
and the recognition of Andria, King of Darien.”
“Don Andria,” the priest said. “Ah? Don Andria, the King. And so
we are not sacked.”
“Now let us find a spade.”
They searched for half an hour before they found a spade.
“We do not use spades,” the priest said. “We tickle the earth with
our toes and it laughs fruits for us.”
“I will lay him here, West,” Margaret said. “Looking towards
England.”
“Sir,” the priest said, “he was a good Catholic. He must look
towards the east.”
“Ah,” said Margaret. “And his wife would like that?”
“Yes. Ah, his wife, sir. Poor child. She was only married six
weeks.”
“It is sad for her. He did not suffer much, father?”
“Ill for four days. But yesterday he was better. Then the fever grew
again. As it does, sir, in some cases. The blood was before the
dawn. Like a child, sir. And his eyes turned upon the Cross.”
“Then I will dig the grave here,” Margaret said. “This will be east
and west by my watch.” He scratched a narrow oblong with the point
of the spade.
“I’ll dig the grave here,” he said.
“Not you. Not in the sun,” the priest said. “There is very much
sickness. Your men will dig.”
“I shall dig,” he answered. He felt that he was burying a part of
Olivia. He would do her that service. He would make a grave for that
unworthy part of her. That act of his should be a part of his penance
towards the dead man’s ghost.
“It is very bad to dig this ground,” the priest said. “It is dust of the
dead. We do not dig deep except for an Excellency. You see. The
rats. Why toil, since God will bring them together at the
Resurrection?”
“This is an evil country,” Margaret answered, driving the spade into
the earth.
“There is fever and death. Very evil,” said the priest. “It is not
wholesome to be in the sun, turning the earth, before chocolate. I will
go to the widow.”
He left the cemetery, holding a handkerchief across his nose. The
rats in their burrow-mouths watched him. One or two of them
scuttled to other burrows. They seemed to play a game of general
post, with Margaret as the “he.”
“Let me have a go at that spade, Captain Margaret, sir,” said West.
“No. I must do this,” he answered. “It is dangerous at the top.
Perhaps deeper down you shall give me a hand. Gather stones from
the wall. I want you to keep away from me, West. This soil is full of
infection. Here is some tobacco. I want you to smoke, all the time
you are here.”
“No, sir,” said West, looking uncomfortable. “Not just now, thank
you, sir.”
“It will keep away the infection. You do smoke?”
“Yes, sir. But it wouldn’t be right, sir, nor respectful to Mrs.
Stukeley.”
“Ah,” said Margaret, feeling himself rebuked. He dug for a few
minutes in silence; it was light, sandy earth, easily shovelled.
“I wish you’d let me do that, sir,” said West.
“No. Not yet, West.”
“He was a big man, too, sir. He’d ’ave been a fine big man if he’d
taken care of himself.”
“Yes,” Margaret answered. “He was a beautiful figure.”
“There’s a lot of poor fellows killed, sir,” said West. “But somehow
it don’t come ’ome like this one does. That yellow look, after what he
was. And the Spanish lady. She wasn’t his real lady, sir.”
“No. She wasn’t his real lady.”
So they talked as they dug turn and turn about for an hour and a
half, when they had to stop digging. They were coming to water. The
bottom of the grave was an inch or two deep in water.
“We can go no further,” Margaret said. “Now we’ll get stones.”
They lined the bed of the grave with stones, and turned to the house
for the body.
The priest and the woman had laid the body out. Margaret ripped
down one of the long, jalousied shutters for a bier, while West
searched in the patio for rope. An old Spanish tapestry of the death
of Absalom served as a pall. They carried Stukeley to the grave, the
priest preceding them, intoning the burial service. Then, very
reverently, they lowered him. West and Margaret went aside after
this and gathered a heap of lilies while the woman and priest prayed
together.
“Take her away, father,” Margaret whispered. The priest led the
woman to the house. She walked like one stunned. Margaret and
West leaned over the grave, to look their last on Stukeley. They
could see the water soaking into the linen, and above that the frayed
body of Absalom, the handsome youth, caught in the thicket, as he
rode. The town was noisy beyond them, two hundred yards away.
Singing and shouting came from the Plaza. It seemed to Margaret to
be the dirge the man would have chosen, this singing and shouting
of men.
“Is that gentleman’s service enough, sir?” West asked.
“Quite enough,” he answered. “You heard him say that Mr.
Stukeley had changed his religion?”
“I’d feel easier if you’d say a few words, sir.”
He spoke the few words. Then with the shovel he began to fill in
the grave, from the foot. He placed many heavy stones among the
earth, so that the rats might be foiled. When he had levelled the
surface he heaped a cairn of stones at the head, and laid the lilies
there. He went to a neighbouring garden, and dug up an arnotto
rose-bush, to plant upon the grave. When this had been planted, the
rites were over, he could do no more. They stood looking down at
the grave for a moment, before they left the graveyard. The singing
was loud behind them. In front of them, darkening under the breeze,
was the bay, with the ships and sloops running in, distant some two
or three miles.
“Come, West,” Margaret said. “I must see after the wounded.” He
took a last look at the grave, at the already drooping lilies and
dejected rose-bush. “Good-bye, Stukeley,” he murmured. He
stooped, and picked some rose-buds, and a little scrap of stone from
the grave, putting them carefully in a pocket-case. “Now smoke,
West,” he said. “And rub tobacco on your hands.”
He reproached himself for having neglected his wounded for so
long; but he knew that Tucket and Pain would look to them. He
wished that the singing and shouting would stop. “Old Rose” and
“The green grass grew” were not songs which the army in his mind
had sung. Thermopylæ was not possible to an army which sang
such songs. Pistol-shots marked the singing of each stanza; there
were yells and cries. He thought he heard the screams of women.
He saw two men come from a house with their arms full of plunder.
“My God,” he said to himself bitterly. “Is Stukeley to check me even
in death?” He drew his pistols again. “Come on, West,” he cried. He
ran to the two looters.
“What are you doing?” he said. “Take that stuff back where you got
it.”
“Get ter hell,” said the men. “You ain’t our captain. Who in hell are
yer talking to?” Both men were drunk. One of them had been
wounded in the head.
“I been doing your dirty work all morning,” said the wounded man,
with drunken gravity. “I’m a free man, and I’m getting a little for
myself. You ain’t my cap. Wot d’yer talk to us for? Go and see ’em in
the Plaza,” said the other man. “Git ter the swamps and shove yer
’ed in.” They rolled off shouting. The noise in the Plaza became
louder at each moment. It was useless to shoot the looters; two-
thirds of the whole force were looting. Pain himself was looting.
Indeed, the sight of the Plaza haunted Margaret like a nightmare
till he died. Of the two hundred men gathered there, hardly thirty
were sober. These stood aloof under Tucket, guarding the wounded
and laughing at the antics of the rest. A heap of loot was piled under
the cotton tree. At every moment a buccaneer added to the pile.
Wine casks lay open about the square, with drunken men lying near
them, in the sun, too drunk to stir. Other drunkards, with linked arms,
danced and sang, making catcalls and obscene noises. A half-
conscious girl lay against a wall, gasping, shaken by hysteria. Her
wild eyes were hard and dry, her hands clutched the dress across
her bosom. Parties of drunken men hacked at doors with axes, and
tossed household gear through the windows on to the heads of other
drunkards beneath. Some had been torturing a Spaniard with
woolding. The man lay dead with the cords about him, his face in the
sands. Others, in wantonness, were now firing the church, dancing
obscenely about in the priests’ robes. Women were screaming in an
upper room. A dozen savages pursued one shrieking woman. They
bawled filthy jests to each other as they ran. Margaret stood over
her, as she fell, moaning, unable to run further. He drove the ruffians
back, threatening them with his sword.
“It’s the cap,” they said. “If the cap wants ’er he must ’ave ’er.” He
placed the woman in a house which had been sacked; it seemed the
safest place in that lost city. Pain came by him, drunk, dragging a
silver tray.
“No looting,” the drunkard called. “Strictly college gents. No looting
’tall. None. Won’t have it. No.” He passed on, crying drunken
catcalls. The eastern side of the town, fanned by the breeze, was
fast spreading to a blaze. The dry wood crackled as the flame
caught. The church roof was pouring smoke. Little flames were
licking out from the eaves. “My God. My God,” said Margaret. “And
this is my act and deed. My act and deed.” He went to Tucket, who
stood with the wounded, grimly watching it all. He could not speak.
He could only shake his head, white to the lips.
“How’s this for hell?” said Tucket. “This is Captain Pain.”
“Are the guards at the gates still?” Margaret answered.
“Some of ’em are. Your own men are. Mine are. I never saw men
like these, though.”
“Let’s get the wounded to the boats. How many have we? How
many men are steady still?”
“Sir,” Tucket answered, “I’ve twenty-five men by me. We lost
fourteen killed. There’s nineteen hurt here. That’s fifty-eight. Say
there’s twenty at the gates still. That leaves a matter of a hundred
and seventy like what you see. Get them hurt into the carts, boys,
and start ’em to the boats. Gently does it. That’s you.”
They laid the wounded men in litters and carts, and wheeled them
down gently to the canoas. Margaret walked by the side of the carts,
talking to the men about their wounds, fanning them with his
handkerchief, getting drink for them, wetting their brows with water
whenever they passed a cistern. The ships were then coming to
anchor within half a mile of the town.
“You won’t be long in the boats,” he told the wounded men. “You’ll
soon be in bed on board. You can see the ships. There they are. Do
you see?”
He spoke to Tucket, urging that they should withdraw the guards
from the east and north gates, lest the Spaniards, guessing what had
happened, should attack suddenly, and overpower them.
“I’ve already sent,” said Tucket. “I ain’t goin’ to lose good men
because these swine choose to raise hell. This was Captain Pain’s
piece. The sooner we’re off the better.”
“I’d sound the assembly,” Margaret said. “But the trumpeter’s
drunk.”
“Could we ring the church bell or something, sir?” said one of the
men.
“The church is on fire.”
“Beg pardon, sir. But we’d oughtn’t to wait,” said Tucket. “We’d
ought to get these fellers on board. The sun’s strong. And we got to
make two journeys as it is.”
At the boats they were joined by the north-gate guards, about a
dozen men in all.
“We can only send away six canoas at a time,” Margaret said.
“That’ll mean twenty-four oars. Two wounded men in each canoa.
You can’t put more, comfortably.”
“That’s so,” said Tucket. “Get ’em in, sons. Ask Captain Cammock
to fire guns.”
They manned six canoas, and laid the worst cases in the
sternsheets. The one sober doctor went with them. He was a clever
surgeon, pretty well known all over the Indies as Doctor Glass Case.
He had left England under a cloud; it was not known why. No man
knew his real name.
“Take them aboard the Broken Heart,” Margaret said. “And then
come back for the rest. Tell Captain Cammock how things stand
here.”
The boats shoved off from the shore below the water-gate. Boat-
covers propped on oars made awnings for the wounded. Margaret
and Tucket watched them quartering on each other, stringing out into
line, some of the stroke oars splashing, so as to spatter water, by
request, into the faces of the wounded.
“Gully-shooting,” Tucket said.
“They’re racing,” said Margaret. He thought how strongly these
men resembled boys. They never lost a chance of competing. Now,
in that hot sun, after seven hours of exertion, they were making the
broad oars bend, driving the canoas through it, racing to the ship.
Men came straggling to the water-gate, asking if they were to go
aboard. Margaret and Tucket told them to tend the wounded, while
they returned to the Plaza to try to bring off the rest of the hands.
There was no question of holding the town. The east side was a
roaring bonfire. All that they could hope was that the Spaniards
would not attack.
“There are three garrisons only twelve miles away,” Margaret said.
“Yes,” Tucket answered. “And them we fit this morning must be in
the woods.”
“I thought I could have trusted Pain,” said Margaret. “They would
have ransomed the town. It’s a merchant town. Look at all the
balsam sheds. And now they’ve thrown it away.”
“That was Captain Pain,” said Tucket. “He said as all he wanted
was for you to help him take the town. He’d do the rest, he said. He’s
deceived you, sir. Deceived you all along. He was telling his hands
just now, he was going to seize your ship, as soon as he got the stuff
aboard.”
They were entering the Plaza as Tucket said this. They were just
in time to see the church roof fall in, with a sudden uprush of fire.
Many of Pain’s men were dragging the loot out of the heat. They
were stripped nearly naked. They tossed the heavy silver from hand
to hand. Sometimes a piece was flung at a man’s head, and then a
fight would begin. At least thirty men lay drunk about the square, too
drunk to move. About thirty others formed a rank across the eastern
side, firing and clubbing at the rats which ran from the burning
houses. They turned and fired at the rats which broke through them.
The Plaza hummed with flying bullets. Bullets were chipping the
adobe walls. Splintered tiles of a soft warm redness lay in flakes
below each house. Whenever a rat was killed, the slayers yelled and
screamed, swinging the corpse by the tail, hitting each other in the
face. Tears ran down Margaret’s cheeks. He had never before seen
a sight like this. He had never seen a mob at work. And these were
the men he had led; these were the men who were to found a new
nation with him.
A sudden roar of cannon made them turn to look seaward. The
Broken Heart was wreathed in smoke from a broadside; but as the
smoke blew clear they could see the danger signal; the foretopsail
dropping to the cap, and a red weft dipped at the peak. Two musket-
shots followed from the fo’c’s’le.
“Danger from the south,” said Margaret. “Here are the garrisons.”
Pain lay in the sand, propped against a wine cask, with his hat
tilted over his eyes. Margaret ran to him and shook him. “Up,” he
cried. “Wake up, man.”
A man came running from the south gate shouting, “The Dagoes.
The Dagoes.” He was bleeding from his mouth. He was gasping his
life out as he ran. “Dagoes,” he gurgled. “Dagoes, you.” He stood for
a moment, swaying, pointing half round the compass behind him.
“Right, son,” said Tucket. “Take a rest. You’re hit. Lie down.”
The man stared at him stupidly, groping with his hands. “Take.
These feathers. Off my teeth,” he gasped, and sank forward gently
from his knees, dead.
Tucket kicked Pain savagely.
“Wha’s a marrer? Wharrer hell’s the marrer?” said Pain, struggling
to his feet.
“We’re attacked,” said Margaret, shaking him. “Get your men.
Lord, man, get your men to the south gate.”
“Hands off, you damcarajo,” he answered angrily. “Why the hell
couldn’t you look out? Hadn’t you sense enough to set a sentry? I’m
awake. You Port Mahon fiddler. What in hell are you looking at me
for? Get the men.”
The guns of the Broken Heart opened fire in succession, blowing
white rings over the trucks. Heavy musket-fire was breaking out at
the south gate. Some of the rat-catchers ran towards it.
“Don’t talk to me like that,” said Margaret. “You drunken little boor.
See what you’ve done. You and your gang of thieves. Look at your
work. Look at it. Answer me again and I’ll run you through.”
“Lord’s sake,” Tucket cried. “They’re on to us. Here they are.
Cuidado, sons.”
“I’ll talk t’you later,” Pain said. “Up with her, sons. Rally an’ bust
’em.” He unslung his piece, and ran, followed by the others, to the
south road. They reached the mouth of it in time to see their fellows
scattering towards them from the breach. All the wall was covered
with clambering Spaniards, hundreds of them. They came swarming
on like a wave of bodies, firing and dropping down to load, firing
again, ever firing, till the air was full of fire. Margaret saw privateers
running up to support him. They came from all sides, fifty or sixty
men in all, enough to make a rough double rank across the street.
They made a stand here, at the corner of the Plaza, fighting steadily
and well, but losing heavily. Margaret picked up a gun and fired with
them, praying only that a bullet might find him soon. He had no
thought of anything save that. He had failed. Now he would die
unpitied in a hopeless fight against odds. There were several
hundreds of Spaniards coming up, and the privateers’ ammunition
failed. They were searching the dead for cartridges. Men were
running back to the Plaza to search the drunkards for cartridges. A
few men climbed to the roof of a house, and fired above the smoke
into the enemy. Margaret climbed up with them, so that he might
order the battle. He lay on the tiles with the rest, firing and cheering.
For ten minutes they lay there, firing till they had no more powder.
Then, as the smoke cleared away, he saw that a troop of horse was
coming up. He saw the Indian lances swaying like boat-masts in a
sea. He climbed down from his perch, at that, and gave the word to
retire, fighting, to the boats. He knew that the fight now was only a
matter of moments.
The men fell back, losing heavily. The Spaniards pressed on,
cheering, trying to flank them. At the boats was a mob of flying
drunkards, struggling with the boat-guard. They were trying to get
the canoas for the loot. The beach was littered with loot. A couple of
thousand pounds’ worth of plate was lying in the sands. Seventy or
eighty men were fighting in the water, shouting and damning, tugging
the canoas to and fro. They left the canoas when the fight ranged
down to the beach. They ran to save the plate from the sands, to
snatch the loot from under the feet of the fighters. They shrieked,
with tears in their eyes, to the fighters. They struck at them with fists
and guns and candlesticks, telling them to save the loot, damn them,
save this precious gear, never mind the Dagoes. The fighters struck
back at them, clubbing their guns. That was the end of the fighting.
The ranks broke. Fighters, drunkards, boat-guard, all the wreck of
the force, were jumbled in a mob among the boats, knee-deep in
water. Margaret, Tucket, and a few more, managed to keep clear of
the mob, and fired at the sallyport as the Spaniards pressed through
to end them. Tucket’s mate cooly filled their pockets with cartridges,
helping himself from the men about him. Two canoas upset; a third,
with only three men in her, drew clear and pulled for the ships.
Spaniards were on the walls above them, firing into them from the
platform. The dead and dying men were beaten down and stamped
on by the herd of wild beasts in the water. Margaret was careless
how it ended for him. He had no wish to live. He felt only the horror
of having mixed with men like these, of having led them, of having
soiled his honour for ever with them. His gun was shattered with a
bullet; the wound in his shoulder had broken out again; he could feel
his shirt, sized with blood gluing to his skin. He drew his sword, and
waited, looking at the walls, watching the heads of the Spaniards
showing in glimpses among the smoke. As he looked there came a
rush above him. A yard of the wall shattered into dust with a burst.
Cammock had opened on the town with his broadside. That saved
them.
He was in the water still. Some loaded canoas were pulling clear.
The last canoas were loading. A dozen steady men, calmed by the
gun-fire, were covering the retreat. One by one they climbed into the
canoas, firing over the gunwales, standing up to fire, anxious to have
the last word. A canoa which had pulled clear backed up to
Margaret, and a voice shouted in his ear. Looking round, he saw that
it was Pain, a grimed and bloody scarecrow, still savage with drink.
“You Portuguese get,” the drunkard screamed. “You called me
down just now. Did you, by God. You junk-laid carajo. Now I’m even.
See?” He swung his knobbed pistol on to Margaret’s brow with a
smash, knocking him senseless. “Give way,” he shouted. “Give way.
That’s what I give to college gents what gets gay with Captain Pain.”
When Margaret recovered consciousness he was in the last
canoa, a hundred yards from the shore. One of the Indians was
mopping his brow with water. Tucket, who was steering, was
uncorking a rum-flask with his teeth.
“You’ll be all right,” Tucket said. “Take a rinse of this.”
“I’m all right,” he answered, with a little, hysterical laugh. “Where is
that man? The man who hit me?”
“Gone aboard your ship,” said Tucket.
“My God,” he answered. “Give way then, quick.”
He sat up, fully roused, splashing water over his head. He felt ill
and stupid; but the thought of possible danger to Olivia roused him.
Looking back, he saw Tolu blazing above the palm-tree tops, the
flames sucking at the forest, scorching the boughs. Looking forward,
he saw the Broken Heart, with men struggling on her gangway amid
the flash of pistols.
“Captain Pain,” Tucket said. “Up with her, boys.”
“I cain’t row no harder,” said one of the men petulantly, like a child
about to cry. “I see old Jimmy shot, as I owed the dollar to. It ain’t my
fault, cap.”
“If I put any more weight on,” said another rower, “this oar’ll go in
the slings. It’s got a chewed slug through the service.”
Margaret noticed then, for the first time, that the canoa had a foot
of water in her. There were seven men in her. Tucket, himself, an
Indian, and four rowers, all of them wounded.
“Let me take an oar,” he said. “Give me your oar, bowman. You’re
hit.”
“I ain’t goin’ to lay up,” the bowman answered. “A sailor don’t lay
up, nor he don’t take medicine, not till he’s dying, and then he don’t
need to.”
“Let me double-bank the stroke then.”
“You stay still,” Tucket said. “You been as near it as most. We’re
the last canoa. D’ye know what that means? We five got an upset
boat and righted her. The Spaniards were riding after us finishing the
wounded. Robin there, the Indian, saved you. He swam a matter of
thirty yards with you before we picked him up. Then we’d to lie-to
and bale her out before she sank, with the Dagoes blazing hell at us.
As it is, we’re only crawling.”
“Robin,” said Margaret, “I shall tell Don Toro to call you by my
name.”
The Indian sucked in his golden nose-plate, and cringed upon his
hams, grinning. He was the only happy man in the force, this Indian
“sin razon.” Tucket added to his happiness by hailing him with his
new name, in his own speech of San Blas pigeon, a jumble of
Spanish and Indian, spoken as English.
“You Captain Margaret now, Robin. Sabe? You Capitano. Capitano
sobre tula guannah anivego. Mamaubah. Eh? Shennorung Capitano.
Muchas mujercillas. Eh?”
Five hundred yards further on the rowers had to rest. They
splashed themselves with water, took a drink of spirits, and lay back
in the stern-sheets. They were worn out. Margaret was worn out, too.
He had no strength left in him. He lay back, dully, watching the ship;
noting, though with no intellectual comprehension, an array of men
passing down the gangway, making a great noise. It was still, after
the roar of the battle. The cries and oaths came down the wind to
him, clearly, across the water. He saw three long canoas, full of men,
pull clear from the ship’s side, with one man, not very big, standing in
the sternsheets of the largest, shrilly cursing at the ship. “That’s my
note,” were the only words which he could catch. “That’s my note.”
He wondered whose note and what note it could be. But his head
was reeling; he didn’t really care.
“There’s a woman in the canoa there,” said one of the rowers.
“My God,” he said, rousing up, as the beaten boxer rises, though
spent, at his second’s cheer. “It’s not Mrs. Stukeley? It’s not Mrs.
Stukeley?”
“No, sir,” said a rower. “The other lady.”