Chapter 21

Analysis of Hydroxyproline in Collagen Hydrolysates

Tobias Langrock and Ralf Hoffmann

Abstract
Hydroxyproline (Hyp) is an imino acid post-translationally formed by sequence-specific hydroxylases
in the repeating collagen Gly–Xaa–Yaa triad present in all collagen types of all species. In both Xaa- and
Yaa-positions, Pro is the most common residue, often oxidized to 4-Hyp in the Yaa- and rarely to 3-Hyp
in the Xaa-positions. Here, we describe the qualitative and quantitative analysis of 3- and 4-Hyp-isomers
by separating the free imino acids either with hydrophilic interaction chromatography (HILIC) or after
derivatization with reversed-phase chromatography (RPC). In both cases, the compounds were detected
by electrospray ionization mass spectrometry (ESI-MS).

Key words: Amino acid quantitation, Collagen, Hydroxyproline, Hydroxyproline isomers, Mass
spectrometry, ESI-MS/MS, Hydrophilic interaction chromatography, Amino acid analysis

1. Introduction

Collagens are present in nearly all organs and tissues in mammals

and form a major constituent of skin, bone, tendon, cartilage,
blood vessels, and teeth. They constitute a quarter of the total
protein content. The most abundant collagen types form fibrils
with a 67 nm axial period, but there are also 14 “non-fibrillar”
collagens. All form a tightly packed, right-handed triple-helix
composed of three left-handed polyproline II (PPII)-like helical
chains wound around each other. Gly-residues, with their small
side chain, are present without distortion in every third position of
the repeating sequence motif Gly–Xaa–Yaa (1–3). Both Xaa- and
Yaa-positions contain mostly proline residues and can be hydroxy-
lated in positions 3 or 4, respectively, by hydroxylases (4–8). In all
collagen types 4-Hyp dominates over 3-Hyp, typically by a factor
of ten or more.

Michail A. Alterman and Peter Hunziker (eds.), Amino Acid Analysis: Methods and Protocols, Methods in Molecular Biology,
vol. 828, DOI 10.1007/978-1-61779-445-2_21, © Springer Science+Business Media, LLC 2012

271
272 T. Langrock and R. Hoffmann

Due to the limitations of Edman degradation in general and

tandem mass spectrometry for isomeric and isobaric compounds,
the analysis of Hyp residues still relies mostly on amino acid analysis
(9). Including the stereocentres at the α-C atom and the hydroxy-
lated C-atom, four stereoisomers exist for both 3- and 4-Hyp, i.e.
a total of eight isomers. Although only the trans-isomers are formed
from L-proline in collagen, the other stereoisomers can be formed
during protein aging or acid hydrolysis in 6 M hydrochloric acid at
elevated temperatures (typically above 100°C) and prolonged
hydrolysis times (typically 4–24 h) (10, 11). Here, we describe two
analytical strategies relying on high-performance liquid chroma-
tography (HPLC) coupled online to electrospray ionization mass
spectrometry (ESI-MS). The first technique separates the hydrolysates
directly on a TSK-Gel Amide 80 column using hydrophilic interac-
tion chromatography (HILIC) (12), i.e. a special type of normal
phase chromatography applicable to polar compounds (13).
The second approach relies on derivatization of the protein
hydrolysates with N2-(5-fluoro-2,4-dinitrophenyl)-L-valine amide
(L-FDVA) (14) followed by reversed-phase (RP-)HPLC (15).

2. Materials

Standard laboratory equipment, such as pipettes (1–1,000 μl),

sample tubes (0.5 and 1.5 ml), analytical balance, centrifuge, and
vortexer are needed in addition to the special materials described
below.

2.1. Apparatus 1. An analytical gradient HPLC-system, ideally equipped with an

autosampler, coupled to an UV-detector (to detect FDVA-
derivatized amino acids) and an ESI-mass spectrometer.
2. To accomplish the most sensitive and selective detection, a
tandem mass spectrometer, such as a triple quadrupole, is
highly recommended.
3. A vacuum centrifuge (e.g. SpeedVac) is used to dry protein
samples and hydrolysates.
4. For sample incubation, a thermo mixer is needed to hold 0.5
and 1.5 ml sample tubes.

2.2. Consumables 1. For reversed phase chromatography – an Aqua C18-column

(internal diameter 2.0 mm, length 150 mm, particle size 3 μm,
pore size 12.5 nm; Phenomenx GmbH, Aschaffenburg,
Germany).
2. For HILIC chromatography – a TSK gel Amide-80-column
(internal diameter 2.0 mm, length 150 mm, particle size 5 μm,
pore size 8 nm; TOSOH Bioscience GmbH, Stuttgart, Germany).
 21 Analysis of Hydroxyproline in Collagen Hydrolysates 273

3. For gas phase hydrolysis, we used a simple and inexpensive

setup with glass inserts for sample tubes (0.25 ml, SUPELCO,
Bellafonte, USA) placed into screw cap tubes with rubber seals
(Fischer Scientific, Leicestershire UK).

2.3. Chemicals 1. Use only reagents of the highest purity available, i.e. at least
HPLC grade eluents and analytical grade reagents.
2. For online LC–MS coupling, LC–MS grade solvents, or even
higher purity solvents, are necessary to provide stable spraying
conditions with a low background during the whole gradient.
3. Prepare all solutions with ultrapure water (at least 18 MΩ·cm
at 25°C) free of any organic contaminants that might bind to
the columns or interfere with the UV-detection or mass spec-
trometry (see Note 1).
4. Formic acid should be the highest quality available, i.e. >99%
for UV-detection and analytical grade for mass spectrometry.
5. Use only hydrochloric acid (6 M) specifically sold to conduct amino
acid analysis (e.g. from Sigma-Aldrich, Steinheim, Germany).
6. FDVA is commercially available (Sigma-Aldrich) but can also
be synthesized with reasonable yields and purities (14).

3. Methods

3.1. Gas Phase 1. Dissolve the protein to be analyzed in an appropriate solvent in

Hydrolysis a defined concentration (ideally 1 mg/ml). Some collagens are
water soluble; others have to be digested with collagenase.
2. Transfer the protein solution (150 μl, i.e. 150 μg protein) into
a glass insert. Place the glass insert into a screw cap sample
tube, but do not cap it.
3. Dry the sample in a vacuum centrifuge.
4. Pipet hydrochloric acid (6 M, 300 μl) into the sample tube,
around the glass insert.
5. Screw the cap with the rubber seal onto the tube and close
tightly. For safety reasons, you should place the sealed tube
into a larger container (e.g. centrifuge tube or glass beaker)
and put this into the drying oven. Make sure that no pressure
can build up in the large container and that the tubes are not
turned upside down.
6. Incubate samples at 110°C for 24 h.
7. Take the container out of the drying oven (wear heat protective
gloves and protective glasses) and let the samples cool down to
room temperature.
274 T. Langrock and R. Hoffmann

8. Take the glass inserts and clean all hydrochloric acid from the
outside. To remove acid that might have condensed in the inserts,
re-centrifuge them in the vacuum centrifuge (30 min).

3.2. HILIC–ESI-MS/MS 1. Prepare HILIC eluent A by dissolving ammonium acetate

(see Note 2) (38.54 mg, 0.5 mmol) in water (100 ml). For eluent B, dissolve
ammonium acetate (192.7 mg, 2.5 mmol) in water (400 ml).
2. Titrate both eluents to pH 5.5 with acetic acid.
3. Add acetonitrile to eluents A (900 ml) and B (600 ml).
4. Dissolve the amino acid samples in eluent A. If charged amino
acids, such as Asp and Glu, are also to be analyzed, then use
eluent B as solvent (see Note 3). For Hyp-analysis in proteins,
dissolve the dried hydrolysates in eluent A (75 μl) and inject
10 μl (20 μg of the original protein).
5. Separate trans-4-Hyp, trans-3-Hyp and cis-4-Hyp (see Note 4),
by using the following gradient, at a flow rate of 150 μl/min:
HILIC gradient

Time point (min) Eluent B (%)

0 5
28.5 60
33.5 95
34 5
44 5

6. Depending on the type of your mass spectrometer, chose one

of the following detection modes.
(a) With a single quadrupole MS use the SIR-mode (m/z
132) to detect the isobaric Hyp-isomers and Ile/Leu or
use a scan mode (m/z 70–300) to detect all amino acids.
(b) With a triple quadrupole MS, you may enhance selectivity
and sensitivity with MS/MS experiments. For detection of
Hyp and Ile/Leu, use MRM experiments (m/z transition
132 → 86). In order to detect all amino acids selectively,
apply a neutral loss scan (neutral loss of 46 u), as all amino
acids tend to eliminate formic acid under MS/MS condi-
tions (16). A chromatogram for Hyp and Ile/Leu is
shown as an example in Fig. 1.
7. For quantitative analyses, prepare a standard containing all
amino acids of interest and inject it several times to record the
signal intensities for at least five different amounts ranging
from 0.1 to 10 nmol of each amino acid. To further improve
the accuracy, we add asparagine as an internal standard to the
hydrolysates (see Note 5).
 21 Analysis of Hydroxyproline in Collagen Hydrolysates 275

Fig. 1. Extracted ion HILIC–ESI-MS chromatogram at m/z 132 (hydroxyproline, isoleucine, and leucine). The sample was a
mixture of 19 proteinogenic amino acids, trans-4-L-Hyp, cis-4-L-Hyp, and trans-3-L-Hyp using a neutral loss scan of 46 in
positive ion-mode. All isobaric components were separated on the TSK Gel Amide 80 column with a linear gradient of
5–60% eluent B over 28.5 min at a flow rate of 150 μl/min.

3.3. Pre-column
Derivatization with
N 2-(5-Fluoro-2,4-
Dinitrophenyl)-L-
Valinamide for RP
Chromatography
1. Dissolve FDVA (11 mg, 36.7 μmol) in acetone (1 ml) to obtain
a 36.7 mM solution. Dissolve NaHCO3 (840 mg) in water
(10 ml) to obtain a 1 M solution. Prepare 1 M hydrochloric
acid by diluting the 6 M hydrochloric acid (100 μl) six fold
with water (500 μl).
2. Use a threefold molar excess of FDVA over the amino acids, as
lower multiples will reduce the accuracy. For real samples, these
numbers have to be judged from the individual amino acid
amounts (see Note 6) or calculated from the estimated protein
quantities (see Note 7).
3. In order to analyze Hyp isomers from collagen, dissolve the
dried hydrolysate in water (15 μl). Transfer one third of this
solution (5 μl) to a fresh tube and add water (16 μl) and
NaHCO3 buffer (8 μl, 1 M).
4. Vortex the solution shortly and add the FDVA solution (32 μl)
prepared in step 1.
5. Incubate the samples (90 min, 40°C) and stop the reaction by
adding hydrochloric acid (8 μl, 1 M). Add acetonitrile (200 μl)
and water (731 μl) to obtain a final volume of 1 ml.
276 T. Langrock and R. Hoffmann

3.4. RP-HPLC 1. Prepare eluents by adding formic acid (1 ml) to water (1 L,

eluent A) or acetonitrile (1 L, eluent B).
2. Separate the six Hyp-isomers (see Note 8) with the following
gradient at a flow rate of 200 μl/min:
RP-HPLC gradient

Time point (min) Eluent B (%)

0 20
30 28
45 36
58 46.5
68 65
73 95
83 95
84 20
105 20

3. This gradient was optimized for an Aqua C18-column (column

length 150 mm, internal diameter 2.0 mm, particle size 3 μm,
and pore size 12.5 nm) originally operated with an aqueous
acetonitrile eluent system containing 0.1% trifluoroacetic acid
(TFA) as the ion pair reagent (see Note 9).
4. The amino acids can be detected and quantified with a
UV-detector or a mass spectrometer, but ideally both systems
are coupled online to the HPLC-system.
(a) Record the absorption at a wavelength of 340 nm to detect
all amino acids with a high sensitivity (Fig. 2).

Fig. 2. A mixture of the six relevant hydroxyproline isomers, i.e. trans-4-Hyp, cis-4-Hyp, trans-3-Hyp, cis-4-D-Hyp, cis-3-D-
Hyp, and cis-3-L-Hyp, was derivatized with N 2-(5-fluoro-2,4-dinitrophenyl)-L-valine amide and separated by RP-HPLC
(C18-Aqua column, absorbance recorded at 340 nm). Eluents were water (A) and acetonitrile (B), both containing 0.1%
formic acid. The gradient used linear increments from 20 to 28% eluent B over 30 min, to 36% eluent B over 15 min, to
46.5% eluent B over 13 min, and to 65% eluent B over 10 min.
 21 Analysis of Hydroxyproline in Collagen Hydrolysates 277

Fig. 3. Extracted ion chromatogram for the N2-(5-fluoro-2,4-dinitrophenyl)-L-valine amide (FDVA) derivatives at m/z 412
(Hyp, Leu, and Ile) from the hydrolysate of the organic matrix of demineralized glass sponge spiculae. The high content
of trans-3-Hyp (more than 20% of the total hydroxyproline) is characteristic for sponge collagen. The detected 4-cis-D-
hydroxyproline most likely results from epimerization during acid hydrolysis.

(b) Use the full scan mode from m/z 300–800 to detect all
derivatized amino acids, including the doubly labelled
lysine. For best quantification, extract the chromatogram
at m/z 412, which corresponds to derivatized Hyp, Ile
and Leu (Fig. 3), or record only an SIR at m/z 412 (see
Note 10). All Hyp-isomers, Ile and Leu can be directly
quantified from this data set.

4. Notes

1. Reliable detection and, in particular, quantification, by LC–MS

demands solvents of special purities. Even solvents sold for
far-UV-applications in chromatography do not usually meet
the high standards required for LC–MS coupling, as they yield
a high chemical background, reducing the sensitivity of the analysis
and contaminating the ionization source and the first section
of the mass spectrometer.
2. Underivatized amino acids are retarded on the Amide-80 column
in HILIC-mode and can be eluted by increasing the polarity of
the eluent, e.g. by increasing the water content. As most amino
acids have very low extinction coefficients in the UV region
above 210 nm, alternative detectors have to be used, such as an
online coupled mass spectrometer. Therefore, only isobaric
amino acids have to be separated by HILIC. Cysteine was not
included in the method development due to its fast oxidation
and dimerization. Basic amino acids (His, Arg, and Lys) are
eluted at high water contents (>50%), but the broad peaks are
difficult to quantify and often show a strong tailing. All other
proteinogenic amino acids can be analyzed by HILIC–ESI–MS,
as the isomeric amino acids (Ile/Leu) and amino acids with similar
masses (e.g. Asn/Asp and Gln/Glu) are well separated.
278 T. Langrock and R. Hoffmann

3. Asn, Asp, Gln, and Glu are not very soluble at high acetonitrile
concentrations. Therefore, these amino acids were dissolved in
eluent B, which can negatively affect the peak shape and chro-
matographic performance of early eluting amino acids, such as
peak-broadening and in severe cases even peak-splitting.
4. All α-L-amino acids racemize during acid hydrolysis at the low
percent level, forming the corresponding α-D-amino acids.
Hydroxyproline with its two stereocentres can thus form epimers,
this means that trans-4-L-Hyp, for example, epimerizes to cis-
4-D-Hyp (11). As trans-4-Hyp is the main Hyp-isomer present
in collagen, a significant amount of cis-4-D-Hyp will be formed
during hydrolysis. The content of cis-4-D-Hyp is at the same
level as trans-3-L-Hyp. It is important, therefore, that this
epimer is separated in HILIC–ESI-MS. As the Amide-80 phase
is non-chiral, HILIC cannot resolve cis-D-Hyp and cis-L-Hyp.
5. The limit of quantification and the linear range both strongly
depend on the mass spectrometer used. The amino acid concen-
trations in collagen hydrolysates will typically range over more
than two orders of magnitude. We found a linear quantification
between 100 pmol and 10 nmol, with correlation coefficients
better than 0.99. Relative standard deviations (RSD) between
multiple injections were below 20%, which is still within an
acceptable range for MS detection and can be further improved
by using internal standards. We recommend Asn as an internal
standard for collagen analysis, as it is not present in protein hydro-
lysates and the RSD is typically better than 10%. Therefore,
dissolve Asn in water (100 pmol/μl) and use this solution to
dissolve the collagen hydrolysate. Using this protocol, 1 nmol of
Asn will be present in the injected hydrolysate (10 μl) as an internal
standard. It is not unusual that amino acids show a non-linear
response in ESI-MS. In such cases, a quadratic regression may
be favourable.
6. To prepare standard solutions of derivatized amino acids, use
stock solutions of 100 mM. Dilute an aliquot of the amino acid
stock solution (10 μl) with water (40 μl) and add first NaHCO3
buffer (20 μl, 1 M) and then FDVA solution (82.5 μl,
36.7 mM). Incubate on a shaker (90 min, 40°C) and neutralize
the mixture with hydrochloric acid (20 μl, 1 M). Add acetonitrile
(200 μl) and water (627.5 μl) to obtain a 1 mM solution of the
derivatized amino acid (1 nmol/μl).
7. In order to derivatize the amino acids with the ideal three
molar excess of FDVA, some assumptions for the derivatization
of protein hydrolysates have to be made. As 150 μg of collagen
are hydrolyzed and dissolved in 15 μl, the 5 μl to be derivatized
contain 50 μg of protein. Assuming a molar mass of 100,000 g/
mol for collagen, this amount reflects 0.5 nmol of protein.
Assuming further, that a collagen molecule contains around
 21 Analysis of Hydroxyproline in Collagen Hydrolysates 279

800 residues and is quantitatively hydrolyzed, 400 nmol of amino

acids have to be derivatized. This requires 1,200 nmol FDVA,
which corresponds to 32 μl of the FDVA stock solution.
8. A complete analysis of all Hyp-isomers potentially present in
collagen hydrolysates demands the separation of at least six
Hyp-isomers. Besides the four L-Hyp isomers potentially present
in collagen, i.e. trans-4-, trans-3-, cis-4- and cis-3-Hyp, cis-4-D-,
and cis-3-D-Hyp have to be separated as well, as these epimers
are formed at a lower percentage from the corresponding
trans-L-isomers during acid hydrolysis. Usually, this epimeriza-
tion is accepted as an error in amino acid analysis. However, to
be able to analyze existing low abundant cis-Hyp in collagen at
exactly this level of concentration, these possible interferences
must be eliminated.
9. During the optimization of the eluent conditions, we also used
TFA instead of formic acid. When the first Aqua C18-column
was replaced by a new column, we realized that the trans-3-
Hyp and cis-4-Hyp was only partially resolved. After flushing
the column overnight with a solution containing 1% TFA,
however, the separation was significantly improved, providing
a baseline separation of both Hyp-isomers with the standard
gradient in the presence of formic acid. This elution profile was
stable afterwards.
10. For additional confidence or enhancement, the described
RP-HPLC-method can be easily coupled online to a mass
spectrometer. Only MS-experiments are useful, as the deriva-
tized amino acids show a complex fragmentation pattern which
hampers the detection in MS/MS-modes. Depending on the
instrumental setup, MS-detection can improve the sensitivity
tenfold compared to UV-detection.

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