European Journal of Pharmaceutical Sciences 45 (2012) 201–204

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European Journal of Pharmaceutical Sciences

journal homepage: www.elsevier.com/locate/ejps

Simultaneous determination of alkyl mesilates and alkyl besilates in finished

drug products by direct injection GC/MS
Uwe Wollein, Nicholas Schramek ⇑
Bavarian Health and Food Safety Authority, Veterinärstr. 2, 85764 Oberschleißheim, Germany

a r t i c l e i n f o a b s t r a c t

Article history: We report a specific, fast and feasible method for the simultaneous determination of methyl mesilate
Received 19 July 2011 (MMS), ethyl mesilate (EMS), isopropyl mesilate (IMS), methyl besilate (MBS) and ethyl besilate (EBS)
Received in revised form 19 September in finished drug products by GC/MS. Sample preparation was carried out by liquid extraction. The ana-
2011
lytes were directly injected into the chromatographic system and quantified with the internal standard
Accepted 9 November 2011
method using methyl tosylate (MTS) as internal standard (ISTD). The method gives excellent sensitivity
Available online 17 November 2011
for all the alkyl and aryl esters at typical target analyte level, according to the acceptance criteria that are
described in the Guideline on the Limits of Genotoxic Impurities which has been issued in 2007 by the
Keywords:
Alkyl mesilates
European Medicines Agency (EMA). The average recovery for methanesulfonic acid esters (mesilates)
Alkyl besilates was not lower than 71 %, for benzenesulfonic acid esters (besilates) not lower than 94%. A linear range
GC/MS with R2 P 0.9998 has been established for concentrations between 0.01 and 1.33 lg/ml. Validation of
Genotoxic Impurities the method was carried out on a sample matrix containing MMS, EMS, IMS, MBS and EBS at relevant lev-
Liquid–liquid extraction els and was further confirmed on finished products containing APIs as mesilate salts (Bromocriptine mes-
ilate, Doxazosin mesilate).
Ó 2011 Elsevier B.V. All rights reserved.

1. Introduction derivatisation procedure recently has been published as a compen-

Genotoxic impurities were strongly discussed in the recent past
(Elder et al., 2008). They may contain functional moieties that
could induce various carcinogenic mechanisms. These overall im-
pacts were recently summarised in order to have a broad overview
from regulatory requirements to analytical control of genotoxic
impurities (Giordani et al., 2011). In each literature about geno-
toxic impurities, sulfonate esters play an important role. Sulfonic
acid esters can occur during drug synthesis, e.g. as counterion in
order to generate a drug salt, as reaction catalyst or protecting
group in a certain reaction step. Furthermore, traces of low molec-
ular alcohol impurities (e.g. methanol, ethanol) deriving from pro-
cess relevant solvents may lead to these sulfonate esters, in
presence of the appropriate alkyl-/arylsulfonic acid or their corre-
sponding acid chloride. The toxicity of alkyl sulfonates is widely
discussed, especially with focus on their DNA alkylating capabili-
ties (Stopper and Lutz, 2002; Gichner, 2003). Even more, many
analytical procedures can be found about the determination of al-
kyl mesilates and/or alkyl besilates in APIs ranging from facile sam-
ple preparations as discussed by Ramjit et al., (1996), they applied
the analytes directly to the GC/MS system, to high sophisticated
analyte derivatisation procedures (Alzaga et al., 2007). A similar
⇑ Corresponding author. Tel.: +49 9131 6808 5451; fax: +49 9131 6808 5838.
E-mail address: nicholas.schramek@lgl.bayern.de (N. Schramek).
dial method on the determination of ‘methyl, ethyl and isopropyl
methanesulfonates in active substances’ (2.5.38) in the European
Pharmacopoeia, supplement 7.3 (publication date 1st of July
2011, implementation date 1st of January 2012). In contrast, a limit
test on APIs was accomplished by Colón and Richoll (2005), who
developed a solid-phase micro extraction (SPME) procedure of
MMS and EMS, before quantitation was carried out by GC/MS. An-
other approach, using LC/MS methodology was investigated by
Taylor et al. (2006).
The maximal daily intake of such impurities is nowadays well
regulated through the Guideline on the Limits of Genotoxic impu-
rities (effective from January 2007) (EMEA/CHMP/QWP/251344/
2006: GUIDELINE ON THE LIMITS OF GENOTOXIC IMPURITIES,
2006) and the follow-up Question & Answer document, which
was last updated in December 2009 (Q&A Clarification Guidance
EMEA/CHMP/SWP/431994/2007 Revision 2, 2009). But as a matter
of fact, this Guideline is not applied retrospectively, thus it gives no
framework to drug products including generics, which received
market authorisation before the Guideline becomes applicable. In
contrast to the mentioned analytical methods on drug substances,
there is poor information for official medicinal control laboratories
or pharmaceutical companies (e.g. small companies) about the
testing on these alkyl mesilates and/or alkyl besilates in finished
drug products (Wollein and Schramek, 2011), in order to prevent
incidents as the Viracept case in 2007 (Elder and Harvey, 2010).

0928-0987/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejps.2011.11.008
202 U. Wollein, N. Schramek / European Journal of Pharmaceutical Sciences 45 (2012) 201–204
Fig. 1. Chemical formulas of alkyl sulfonic acid esters MMS (R1 = CH3), EMS
(R1 = CH2CH3), IMS (R1 = CH(CH3)2), aryl sulfonic acid esters MBS (R2 = CH3, R3 = H),
EBS (R2 = CH2CH3, R3 = H) and internal standard MTS (R2 = CH3, R3 = CH3).
Regarding these facts and the non-retrospective application of the
EMA Guideline, it seems necessary to develop one practicable
method for the determination of both alkyl- (MMS, EMS, IMS,
Fig. 1) and arylsulfonates (MBS, EBS, Fig. 1) in drug products with
facile sample workup and direct injection to a GC/MS system.
2. Experimental
2.1. Chemicals
Methyl mesilate, ethyl mesilate and methyl tosylate were pur-
chased from Sigma Aldrich (Taufkirchen, Germany), isopropyl mes-
ilate from Acros (Geel, Belgium). Methyl besilate was supplied
from Alfa Aesar (Karlsruhe, Germany) and ethyl besilate from ABCR
(Karlsruhe, Germany). For liquid–liquid extraction n-Hexane Pest-
analÒ, solvent for residual analysis was obtained from Fluka (Buc-
hs, Switzerland).
2.2. GC/MS conditions
A Shimadzu GC/MS QP-2010 plus, equipped with an combi-PAL
autosampler (AOC 5000) for liquid and static headspace injection
(Kyoto, Japan) was used. Chromatographic separations were
achieved on a Restek Rxi 5-Sil MS (30 m  0.32 mm, 0.5 lm film
thickness) capillary column (Bellefonte, USA). The column temper-
ature was set to 80 °C for 1 min, programmed to 300 °C at 12 min 1
and held at this final temperature for 5 min. The column flow using
helium as carrier gas was held constant at 1.5 ml min 1. Injector
temperature was 250 °C set at split mode (20:1) with an injection
volume of 1 ll. The detector was operated with electron impact
ionisation (EI, 70 eV) with an ion source temperature of 230 °C
and an interface temperature of 280 °C. Ions were acquired in sin-
gle ion monitoring (SIM) mode with a solvent cut time of 2.0 min.
The SIM parameters were set at m/z 80, 65, 95 for MMS, m/z 79, 97,
109 for EMS, m/z 123, 43, 79 for IMS, m/z 77, 141, 172 for MBS, m/z
Fig. 2. Typical chromatogram of a spiked sample matrix preparation. Matrix was spiked with 0.7–1.0 lg ml
3.6 min; MBS, RT 8.4 min; EBS, RT 9.2 min) and the internal standard (MTS, RT 9.8 min).
77, 51, 141 for EBS and m/z 91, 155, 186 for MTS with each first as-
signed ion used for quantification.
2.3. Preparation of standard and sample solutions
The stock solution containing MMS, EMS, IMS, MBS and EBS was
prepared at approximately 150 lg ml 1 each in n-hexane. This
solution can be used for a maximum of 72 h when stored in a
refrigerator. Additionally a stock solution containing MTS as ISTD
was prepared in a similar manner, but with a concentration of
approximately 300 lg ml 1.
For linearity validation the stock solution containing the three
mesilates and two besilates was diluted with n-hexane to give con-
centrations at 0.01, 0.1 and 1 lg ml 1. To each of the concentra-
tions, prepared in a 10 ml volumetric flask, 40 ll of the ISTD
stock solution was added and filled up to volume with n-hexane,
to contain about 1.2 lg ml 1 ISTD. These dilutions have to be pre-
pared freshly before use.
For sample preparation, the representative sample matrix (con-
taining equal amounts of lactose and microcrystalline cellulose) or
the solid dosage form of the finished drug product, conscientiously
crushed in a mortar, were weighed. The amount of matrix to be ta-
ken should contain about 25 mg of the API. The maximum weight
may vary, depending on the average weight of the dosage form, but
should not exceed 1.0 g. The fine powder is transferred in a silica
glass centrifuge tube with a PTFE screw cap and suspended in
4.0 ml of n-hexane, after adding of 20 ll of ISTD stock solution
(concentration of ISTD in the sample solution: 1.5 lg ml 1). The
closed tube was shaken horizontally for 15 min and then centri-
fuged at 4000 rpm. One microlitre of the clear supernatant was
immediately applied to GC/MS analysis. It should be noted, that
each sample preparation should be measured within 3 h.
For method recovery tests, each sample was prepared following
the procedure described above, but 30 ll of the standard stock
solution and 20 ll of the ISTD stock solution were added before li-
quid extraction. Fig. 2 shows a typical chromatogram of a sample
matrix spiked with MMS (RT 2.6 min), EMS (RT 3.2 min), IMS (RT
3.6 min), MBS (RT 8.4 min), EBS (RT 9.2 min) and the internal stan-
dard MTS (RT 9.8 min).
3. Results and discussion
Aim of this work was the development of a method, which is
appropriate for both, mesilates and besilates in solid dosage drug
products. We found GC/MS to be the most practical when mesi-
lates should be detected in parallel with besilates. Direct injection
may be preferred as the sulfonic acid esters are measured directly
and quantitation is not influenced as it may occur, when a deriva-
tization step is inserted as recommended by literature (Alzaga
1
of each analyte (MMS, RT 2.6 min; EMS, RT 3.2 min; IMS, RT
 U. Wollein, N. Schramek / European Journal of Pharmaceutical Sciences 45 (2012) 201–204 203

et al., 2007) or the compendial method. For quantitation, the use of Table 2
a stable internal standard with similar chemical properties and Survey of the results from the validation of mesilates and besilates in sample matrix.

similar retardation factor compared to the analytes of interest is Analyte Linearity LOQ (lg/ LOD (lg/ Average recovery RSD
necessary. Methyl tosylate was found to fulfil all these criteria. Be- ml) ml) (%) (%)
cause of the bulky powder that has to be weighed for the analysis MMS 0.99987 0.017 0.005 82.41 7.12
of drug products, liquid extraction of the sulfonate esters was the EMS 0.99981 0.014 0.004 96.25 3.40
method of choice. The best solvent for this extraction technique IMS 0.99987 0.012 0.004 91.21 3.41
MBS 0.99996 0.010 0.003 101.76 2.62
had to be found and our efforts resulted in n-hexane. It is worth EBS 0.99991 0.013 0.004 100.97 1.66
mentioning that application of n-hexane gives a huge benefit in li-
quid extraction: only spare amounts of API or possible present fur-
ther impurities are extracted from the matrix, as high amounts of
API or impurities may dramatically affect the limit of detection described under 2.3 and showed recovery from 72–95% (Table 1).
(LOD) or detection selectivity via ion depression. Moreover, stabil- Similar results were achieved in testing neat APIs (Bromocriptine
ity of the analytes of interest is assured over a longer period in- mesilate, Doxazosin mesilate and Trimipramin mesilate).
stead of using solvents of higher polarity or with nucleophilic
properties. A big drawback using n-hexane is that the method is 3.1. Linearity of the esters
not suitable for liquid dosage forms because of the low polarity
of the solvent versus the high polarity of the sulfonic acid esters The linearity of the five sulfonic acid esters was satisfactorily
since liquids are almost ever composed of water or ethanol–water
demonstrated with correlation coefficients >0.9998 and is summa-
mixtures. rised in Table 2. Since there is no need of a wide calibration range
In contrast to the method 2.5.38 ‘methyl, ethyl and isopropyl
only a three point calibration graph (between 0.01 and 1 ll min 1)
methanesulfonate in active substances’ described in the European was chosen.
Pharmacopoeia supplement 7.3, the method described in here is
aimed at solid dosage forms of finished drug products and needs
no further derivatization steps of the analytes. It may also serve 3.2. Recovery of the esters
as alternative on APIs, which was tried out during a market surveil-
lance study arranged by the EDQM. The studies outcome was not The accuracy was demonstrated by the recovery of the five es-
taken into account during this validation process, but it provides ters from the sample matrix spiked with the standard stock solu-
results in a satisfactory manner. tion containing about 0.7–1.0 lg ml 1 of each analyte and the
According to the threshold of toxicological concern (TTC) value internal standard, respectively. Nine separate sample preparations
of the Guideline on the Limits of Genotoxic Impurities (EMEA/ were injected within 3 h after preparation to give recoveries from
CHMP/QWP/251344/2006: GUIDELINE ON THE LIMITS OF GENO- 72–93% (average: 82.41%) for MMS, 86–102% (average: 96.25%) for
TOXIC IMPURITIES, 2006) we set the lowest calibration level at EMS, 79–99% (average: 91.21%) for IMS, 95–106% (average:
0.01 lg ml 1, corresponding to 0.04 lg g 1 tablet or capsule pow- 101.76%) for MBS and 94–104% (average: 100.97%) for EBS, respec-
der since this is a 37.5-fold lower amount as the TTC of 1.5 lg per- tively (Table 2). The precision was expressed by the relative stan-
mitted per person per day. dard deviation (RSD). The demand was to achieve a RSD value
With the parameters in hand we directed our attention in test- <5%, MMS excluded. The higher RSD value of 7.1% is influenced
ing of authorised drug products. The samples were obtained from by the low concentration of the analyte and a slight carry-over ef-
wholesalers in the course of our yearly sample plan. The supplied fect that can be diminished through an injection of blank n-hexane
drug products contained APIs as Bromocriptine mesilate and Dox- after each second standard and sample run. The assigned uncer-
azosin mesilate. Sample analysis showed no presence of any mes- tainty of measurement is lower than 3.0% (confidence level
ilate. The samples were spiked corresponding to the procedure p = 0.95) for each sulfonate.

Table 1
Results and recovery of the testing of two authorised drug products containing an API as mesilate salt (ppm specification is due to the different sample weight).

Injection # ppm (actual result) ppm (spiked sample) ppm (target value) Recovery %
Analyte: Bromocriptin capsules
(1) MMS <LOD 4.7 6.4 73.44
EMS <LOD 5.5 7.1 77.46
IMS <LOD 5.0 5.5 90.91
(2) MMS <LOD 4.7 6.4 73.44
EMS <LOD 5.6 7.1 78.87
IMS <LOD 5.1 5.5 92.73
Average recovery %
MMS 73.44
EMS 78.17
IMS 91.82
Analyte: Doxazosine mesilate tablets
(1) MMS <LOD 3.1 4.3 72.09
EMS <LOD 3.8 4.8 79.17
IMS <LOD 3.3 3.7 89.19
(2) MMS <LOD 3.5 4.3 81.40
EMS <LOD 4.1 4.8 85.42
IMS <LOD 3.5 3.7 94.59
Average recovery %
MMS 76.74
EMS 82.29
IMS 91.89
204 U. Wollein, N. Schramek / European Journal of Pharmaceutical Sciences 45 (2012) 201–204
3.3. Limit of detection (LOD) and limit of quantification (LOQ)
The LOD and LOQ were calculated from S/N data generated from
three injections of a mixed standard (in absence of an API) with a
concentration of approximately 0.1 lg ml 1 of each ester. For each
sulfonate LOQ is included in the calibration curve. Splitless injec-
tion did not have a decreasing effect on both, LOD and LOQ (Table
2).
4. Conclusion
A general GC/MS methodology using liquid extraction and di-
rect injection has been developed for the determination of geno-
toxic alkyl and aryl sulfonic acid esters in finished drug products.
It is suitable for the testing of already (long) licensed products.
The method shows good linearity over the relevant range that
should be focused when testing according to the TTC value which
is introduced by the EMA Guideline on the Limits of Genotoxic
Impurities. The unpolar solvent n-hexane provides major advanta-
ges in inhibiting API extraction and assures longer stability of the
analytes. The method could be transferred to analysis of authorised
products with satisfactorily recovery at relevant levels. Some pre-
caution should be taken due to the determination of methyl mes-
ilate as it might show little carry-over in the GC/MS system.
Acknowledgement
We thank Rosalia Spirkl for her technical assistance.
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