Gc-ms studies on derivatization of creatinine and creatine by bstfa and their measurement in human urine

Article abstract of DOI:10.3390/molecules26113206

In consideration of its relatively constant urinary excretion rate, creatinine (2-amino-1methyl-5H-imidazol-4-one, MW 113.1) in urine is a useful endogenous biochemical parameter to correct the urinary excretion rate of numerous endogenous and exogenous substances. Reliable measurement of creatinine by gas chromatography (GC)-based methods requires derivatization of its amine and keto groups. Creatinine exists in equilibrium with its open form creatine (methylguanidoacetic acid, MW 131.1), which has a guanidine and a carboxylic group. Trimethylsilylation and trifluoroacetylation of creatinine and creatine are the oldest reported derivatization methods for their GC-mass spectrometry (MS) analysis in human serum using flame-or electron-ionization. We performed GC-MS studies on the derivatization of creatinine (d₀-creatinine), [methylo-²H₃ ]creatinine (d₃ creatinine, internal standard) and creatine (d₀-creatine) with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) using standard derivatization conditions (60 min, 60^?C), yet in the absence of any base. Reaction products were characterized both in the negative-ion chemical ionization (NICI) and in the positive-ion chemical ionization (PICI) mode. Creatinine and creatine reacted with BSTFA to form several derivatives. Their early eluting N,N,O-tris(trimethylsilyl) derivatives (8.9 min) were found to be useful for the precise and accurate measurement of the sum of creatinine and creatine in human urine (10 μL, up to 20 mM) by selected-ion monitoring (SIM) of m/z 271 (d₀-creatinine/d₀-creatine) and m/z 274 (d₃-creatinine) in the NICI mode. In the PICI mode, SIM of m/z 256, m/z 259, m/z 272 and m/z 275 was performed. BSTFA derivatization of d₀-creatine from a freshly prepared solution in distilled water resulted in formation of two lMate-eluting derivatives (14.08 min, 14.72 min), presumably creatinyl-creatinine, with the creatininyl residue existing in its enol form (14.08 min) and keto form (14.72 min). Our results suggest that BSTFA derivatization does not allow specific analysis of creatine and creatinine by GC-MS. Preliminary analyses suggest that pentafluoropropionic anhydride (PFPA) is also not useful for the measurement of creatinine in the presence of creatine. Both BSTFA and PFPA facilitate the conversion of creatine to creatinine. Specific measurement of creatinine in urine is possible by using pentafluorobenzyl bromide in aqueous acetone.

Full text of DOI:10.3390/molecules26113206

molecules
Article
GC-MS Studies on Derivatization of Creatinine and Creatine by
BSTFA and Their Measurement in Human Urine
Olga Begou † , Kathrin Weber †, Bibiana Beckmann and Dimitrios Tsikas *
Institute of Toxicology, Core Unit Proteomics, Hannover Medical School, 30625 Hannover, Germany;
mpegolga@chem.auth.gr (O.B.); kathrin.weber89@gmx.net (K.W.); beckmann.bibiana@mh-hannover.de (B.B.)
* Correspondence: tsikas.dimitros@mh-hannover.de
† These authors contributed equally to this work.
Abstract: In consideration of its relatively constant urinary excretion rate, creatinine (2-amino-1-
methyl-5H-imidazol-4-one, MW 113.1) in urine is a useful endogenous biochemical parameter to cor-
rect the urinary excretion rate of numerous endogenous and exogenous substances. Reliable measure-
ment of creatinine by gas chromatography (GC)-based methods requires derivatization of its amine
and keto groups. Creatinine exists in equilibrium with its open form creatine (methylguanidoacetic
acid, MW 131.1), which has a guanidine and a carboxylic group. Trimethylsilylation and triflu-
oroacetylation of creatinine and creatine are the oldest reported derivatization methods for their
GC-mass spectrometry (MS) analysis in human serum using flame- or electron-ionization. We per-
formed GC-MS studies on the derivatization of creatinine (d0-creatinine), [methylo-2H3]creatinine (d3-
creatinine, internal standard) and creatine (d0-creatine) with N,O-bis(trimethylsilyl)trifluoroacetamide
(BSTFA) using standard derivatization conditions (60 min, 60 ◦C), yet in the absence of any base.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Reaction products were characterized both in the negative-ion chemical ionization (NICI) and in the
positive-ion chemical ionization (PICI) mode. Creatinine and creatine reacted with BSTFA to form
several derivatives. Their early eluting N,N,O-tris(trimethylsilyl) derivatives (8.9 min) were found to
be useful for the precise and accurate measurement of the sum of creatinine and creatine in human
Citation: Begou, O.; Weber, K.;
Beckmann, B.; Tsikas, D. GC-MS
Studies on Derivatization of
Creatinine and Creatine by BSTFA
and Their Measurement in Human
Urine. Molecules 2021, 26, 3206.
https://doi.org/10.3390/
urine (10 µL, up to 20 mM) by selected-ion monitoring (SIM) of m/z 271 (d0-creatinine/d0-creatine)
and m/z 274 (d3-creatinine) in the NICI mode. In the PICI mode, SIM of m/z 256, m/z 259, m/z 272
and m/z 275 was performed. BSTFA derivatization of d0-creatine from a freshly prepared solution
in distilled water resulted in formation of two lMate-eluting derivatives (14.08 min, 14.72 min),
presumably creatinyl-creatinine, with the creatininyl residue existing in its enol form (14.08 min)
and keto form (14.72 min). Our results suggest that BSTFA derivatization does not allow specific
analysis of creatine and creatinine by GC-MS. Preliminary analyses suggest that pentafluoropropionic
anhydride (PFPA) is also not useful for the measurement of creatinine in the presence of creatine.
Both BSTFA and PFPA facilitate the conversion of creatine to creatinine. Specific measurement of
creatinine in urine is possible by using pentafluorobenzyl bromide in aqueous acetone.
molecules26113206
Academic Editor: Paraskevas
D. Tzanavaras
Received: 7 May 2021
Accepted: 22 May 2021
Published: 27 May 2021
Keywords: BSTFA; creatine; creatinine; derivatization; quantification; silylation; TMS; validation
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Creatinine (2-amino-1,5-dihydro-1-methyl-4H-imidazol-4-one, MW 113.12; see Scheme 1)
is the end-product of creatine catabolism. Creatinine is excreted in the urine with a fairly
constant rate and is generally used for the correction of renal excretion rates of endogenous
and exogenous substances. This correction is indispensable in clinical studies when urine
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© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
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4.0/).
specimens from spontaneous micturition must be analyzed [
creatinine in urine samples of healthy adults is approximately 12–13 mM, with men excret-
ing higher amounts of creatinine than women [ ]. Besides the spectrophotometric method
based on the famous Jaffé reaction [ ] many different analytical methods are currently avail-
able for creatinine. They include spectrophotometric, enzymatic and instrumental methods
1]. The mean concentration of
1
2
Molecules 2021, 26, 3206. https://doi.org/10.3390/molecules26113206
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 3206
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based on HPLC, GC-MS, LC-MS and LC-MS/MS [3–19]. Lawson [20] and Siekmann [21]
demonstrated by electron ionization (EI) that creatinine reacts with silylation reagents to
form its N,N,O-tris(trimethylsilyl) derivative. Björkhem and colleagues used trifluoroacetic
anhydride for the derivatization of creatinine and its GC-MS analysis [22]. Trimethylsilyla-
tion derivatization reactions used in MS-based methods were found to be associated with
interferences due to formation of several derivatives [23]. To our knowledge, the GC-MS
measurement of urinary creatinine as N,N,O-tris(trimethylsilyl) derivative by negative-ion
chemical ionization (NICI) or positive-ion chemical ionization (PICI) has not been reported
thus far.
Scheme 1. Schematic of the expected derivatization reaction and products of ( ) unlabeled creatinine (d0-creatinine),
A
(
B
) deuterium-labelled creatinine ([methylo-2H3]creatinine, d3-creatinine) and (
C) unlabeled (d0-creatine) and with N,O-
tris(trimethylsilyl)trifluoroacetamide (BSTFA) to form their N,N,O-tris(trimethylsilyl) creatinine derivatives (
N,N,N’,O-tetrakis(trimethylsilyl creatine derivative (C).
A,B) and
In the present study, we investigated in detail the derivatization of unlabelled creati-
nine (d0-creatinine), commercially available [methylo-2H3]creatinine (d3-creatinine) and
unlabelled creatine (d0-creatine) by N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), one
of the oldest trimethylsilylation reagents for amino acids [24]. It is well known that BSTFA
reacts with many functional groups, notably hydroxyl, carboxyl and amine groups. Based
on this knowledge we expected that BSTFA will react with creatinine and creatine to form
N- and O-derivatives (Scheme 1).

Molecules 2021, 26, 3206
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In our study, we used GC-MS in the NICI and in the PICI mode, confirmed the forma-
tion of the expected N,N,O-tris(trimethylsilyl) derivatives and identified several derivatives
of creatinine and creatine that have not reported thus far. As creatinine and creatine are
in a pH-dependent equilibrium and inter-convertible, our results suggest that BSTFA and
GC-MS are not specific for creatinine and creatine but allow measurement of their sum.
Using d3-creatinine as the internal standard we demonstrate that creatinine can be reli-
ably quantitated in 10-µL aliquots of human urine by GC-MS as N,N,O-tris(trimethylsilyl)
derivative with minimum labour.
2. Materials and Methods
2.1. Chemicals and Materials
Unlabeled creatine (d0-creatine), unlabeled creatine phosphate 4 H2O, unlabeled cre-
×
atinine (d0-creatinine) and trideuterocreatinine, i.e., [methylo-2H3]creatinine (d3-creatinine;
declared isotopic purity of >99 atom% 2H) were obtained from Aldrich (Steinheim, Ger-
many). Stock solutions of d0-creatine, d0-creatinine and d3-creatinine (each 20 mM) were
◦
prepared in deionized water and stored in a refrigerator at 8 C. BSTFA was obtained
from Macherey-Nagel (Düren, Germany). Glassware for GC–MS (i.e., 1.5 mL autosam-
pler glass vials and 0.2 mL microvials) and a fused-silica capillary column Optima 17
(15 m × 0.25 mm I.D., 0.25 µm film thickness) were purchased from Macherey-Nagel.
2.2. Derivatization Procedure for Creatinine in Human Urine Samples
Urine samples used in method development and validation were obtained from
healthy volunteers being members of the researcher group and authors of this manuscript.
Urine samples (1-mL aliquots) were kept frozen at
derivatization, urine samples were thawed and centrifuged (5800
and synthetic creatinine-containing samples (usually 10 L) were evaporated to complete
dryness under a stream of nitrogen. Subsequently, the samples were treated with 100
−
18 ◦C until analysis. Prior to sample
×
g, 5 min). Urine (10 µL)
µ
µL
absolute ethanol and the solvents were evaporated to dryness by a stream nitrogen gas
to remove remaining water. Then, the residues were reconstituted with pure BSTFA
(100
After cooling to room temperature, aliquots (about 90
autosampler glass vials equipped with 200- L microinserts. Aqueous solutions (usually
µ
L), the glass vials were tightly closed and heated for 60 min at 60 ◦C in a thermostat.
µL) were transferred into 1.8-mL
µ
10 µL aliquots) of creatinine and creatine were derivatized as described above.
2.3. GC–MS Conditions
In this work, we used a GC-MS method previously used in our group for amino
acid derivatives [25]. GC-MS analyses were performed on a single-quadrupole mass
spectrometer model ISQ directly interfaced with a Trace 1310 series gas chromatograph
equipped with an autosampler AS 1310 from ThermoFisher (Dreieich, Germany). The
following oven temperature program was used with helium as the carrier gas at a constant
flow rate of 1 mL/min: 0.5 min at 40 ◦C, then increased to 210 ◦C at a rate of 15 ◦C/min
and to 320 ◦C at a rate of 35 ◦C/min, respectively, and held at 320 ◦C for 1 min. Interface,
◦
◦
◦
injector and ion-source were kept at 300 C, 280 C and 250 C, respectively. Electron
energy was set to 70 eV and electron current to 50 A. Methane (2.4 mL/min) was used
as the reagent gas for NICI and PICI. Aliquots (1 L from derivatization mixtures) were
injected in the splitless mode by means of the autosampler using a 10- L Hamilton needle,
which was cleaned automatically three times with toluene (5 L) after each injection.
µ
µ
µ
µ
Quantitative analyses were performed in the selected-ion monitoring (SIM) mode. The
peak area (PA) values of d0-creatinine and d3-creatinine were calculated automatically by
the GC–MS software (Xcalibur and Quan Browser). The concentration of d0-creatinine was
calculated by multiplying the peak area ratio (PAR) of d0-creatinine to d3-creatinine with
the concentration of d3-creatinine added to the sample. Statistical analyses and graphs
were performed and prepared by GraphPad Prism 7 (San Diego, CA, USA).

Molecules 2021, 26, 3206
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2.4. HPLC Analysis of Creatine, Creatinine and Creatine-Phosphate in HCl Solutions
We used a HPLC method previously reported by our group for creatinine measure-
ment in human urine [16]. HPLC analyses were carried out on the an HPLC system
consisting of an Agilent 1100 Series binary pump G1312A, an Agilent 1100 Series Degaser
G1322A, an Agilent 1100 Series oven column Colcom G1316A, an Agilent 1100 Series VWD
detector (all Agilent, Waldbronn, Germany, and an MP3 autosampler (Gerstel, Mülheim,
Germany), ChemStation for LC-Systems Rev.B.0402SP1 (212) and Gerstel Maestro Version
1.3.20.41.13.5 were used to control the HPLC system and evaluate the analyses. HPLC
analyses were performed on a Kinetex 5
Phenomenex (Aschaffenburg, Germany) at a fixed column temperature of 20 C. The
mobile phase was 100 mM sodium acetate, pH 7.5, 10 vol% methanol and was pumped
µ
m EVO C18 100 Å column (250 × 4.6 mm) from
◦
at a flow rate of 1.0 mL/min. Samples (20
pler. The effluent was monitored at 210 nm. The analysis time was 5 min. The retention
time was 2.073 0.018 min for creatine-phosphate, 2.252 0.007 min for creatine and
µL) were injected by means of the autosam-
±
±
2.547 ± 0.007 min for creatinine.
3. Results
3.1. Generation of GC-MS Spectra and Characterization of Derivatization Products of d0-Creatine
and of d3-Creatinine
Each 100 nmol of d0-creatinine and d3-creatinine taken from their aqueous solutions
were combined, the solvent was evaporated to dryness and derivatized with 100
as described above. Derivatization resulted in a yellow-colored clear solution. The sample
was analyzed by GC-MS in the PICI and NICI mode consecutively by injecting 1- L aliquots
µL BSTFA
µ
of the BSTFA solutions corresponding each to 1 nmol of d0-creatinine and d3-creatinine
(assuming quantitative derivatization). GC-MS spectra were generated by scanning the
quadrupole in the mass-to-charge (m/z) ratio range of 50–650 and 50–1000 (1 scan per s).
We observed two chromatographic peaks with the retention time of 8.6 min and 8.9 min
(major peak) and their GC-MS spectra contained paired m/z values differing by 3 Da due
to the three deuterium atoms in methyl group of d3-creatinine (Figure 1).
The most intense anions in the NICI mass spectrum (Figure 1A) of the GC peak eluting
at 8.9 min were m/z 271 and m/z 274 (base peaks). Less intense anions were found at
m/z 199 and m/z 202, and very weak ions (intensity < 1%) were m/z 326 and m/z 329,
presumably due to molecular anions of the derivatives (i.e., [M]−). These data indicate
the presence of the unlabeled methyl group in d0-creatinine and of the deuterium-labeled
methyl group of d3-creatinine in this peak (Figure 1A). The NICI spectrum of this GC
peak also contained weak anions at m/z 144 and m/z 186 that do not carry the original
methyl group of creatinine (Figure 1A). The PICI mass spectrum of the GC peak eluting at
8.9 min contained intense cations at m/z 272, m/z 275, m/z 256 and m/z 259, less intense
cations at m/z 300 and m/z 303, weaks ions at m/z 312 and m/z 315, and very weak ions
(intensity < 1%) at m/z 330 and m/z 333, presumably due to the protonated molecules of
the derivatives (i.e., [M+H]+) (Figure 1B).
These data indicate the presence of the unlabeled methyl group in d0-creatinine and
the deuterium-labeled methyl group of d3-creatinine in this peak (Figure 1). Comparison
of the total ion intensity in the NICI and PICI mass spectra (1.85
×
106 versus 9.92
×
105,
Figure 1) suggests that NICI may allow for a more sensitive detection of creatinine than
PICI. Proposed fragmentation mechanisms of the N,N,O-trimethylsilyl derivatives in the
PICI are shown in Scheme 2.
The smaller GC peak eluting at 8.6 min had closely comparable NICI and PICI mass
spectra to those of the N,N,O-tris(trimethylsilyl) derivative (data not shown). These ob-
servations suggest that the GC peak with the retention time of 8.6 min is an isomer of the
N,N,O-tris(trimethylsilyl) derivative of creatinine.

Molecules 2021, 26, 3206
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Figure 1.
(A) Negative-ion chemical ionization (NICI) and (B) positive-ion chemical ionization (PICI) GC-MS
spectra generated from a mixture of d0-creatinine (blue) and d3-creatinine (red) after derivatization with N,O-
tris(trimethylsilyl)trifluoroacetamide (BSTFA) at 60 ◦C for 60 min (each 1 nmol injected). The retention time (tR) of
the GC-MS peak was 8.9 min. Insets indicate the proposed structures of the derivatives and ions. See Scheme 2.

Molecules 2021, 26, 3206
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Scheme 2. Proposed fragmentation mechanisms for the N,N,O-trimethylsilyl derivatives of d0-creatinine (
d3-creatinine ( , red) of the GC-MS peak with the retention time of 8.9 min in the PICI mode. The numbers give the
molecular weight of the neutral substances and the cations. See Figure 1B.
A, blue) and
B
The GC peak with the retention time of 8.7 min was only detectable in the PICI mode.
The PICI mass spectrum of this peak contained three pairs of cations differing by 3 Da
due to the presence of d3-creatinine, i.e., m/z 314 and m/z 317 (base peaks), m/z 330 and
m/z 333 ([M+H]+), and m/z 358 and m/z 361 ([M+C2H4+H]+) (Figure 2). Adducts such as
C2H4 (28 Da) are common in PICI of amines such as dimethyl amine and derive from the
reactant gas methane [26]. Presumably, the adduct is on the non-ring amine group. These
observations suggest the GC peak eluting at 8.7 min is a creatinine derivative with three
trimethylsilyl (TMS) groups, most likely the N2,N3,O4-tris(trimethylsilyl) derivative. It
cannot ionize in the NICI mode, presumably because of the inability to form anions by loss
of an H atom or by capturing an electron due to the lack of electron-capturing atoms and
functional groups in the derivative. The cations with m/z 314 and m/z 317 seem to be very
stable and do not fragment. The cations m/z 55, m/z 57, m/z 73 and m/z 147 are shared by
d0-creatinine and d3-creatinine and are likely to be associated with the TMS groups (see
also [23]) of the derivatives (see also Figure 1B).

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Figure 2. PICI GC-MS spectrum generated from a mixture of d0-creatinine (blue) and d3-creatinine (red) after derivatization
◦
with BSTFA at 60 C for 60 min. The retention time (tR) of the GC-MS peak was 8.7 min. Insets indicate the proposed
structures for the mass fragments. See also Figure 1B.
3.2. Generation of GC-MS Spectra and Characterization of Derivatization Products of d0-Creatine
Derivatization of d0-creatine with BSTFA (60 ◦C, 60 min) resulted in the formation
of three GC-MS peaks with the retention times of the d0-creatinine. The NICI and PICI
mass spectra of these derivatives were virtually identical with those of the d0-creatinine
derivatives (data not shown). In order to investigate the potential formation of additional
derivatives of d0-creatine we extended the upper m/z scanning range to 1000 and the
acquisition time to 16 min. We observed two GC-MS eluting at 14.08 min (minor peaks)
and 14.72 min (major peaks) in the NICI and PICI mode. The corresponding GC-MS spectra
of these d0-creatine derivatives and the relatively high difference in their long retention
times suggest that these peaks are not derivatives of d0-creatine or d0-creatinine (Figure 3).
A possible explanation could be the formation of a creatinyl-derivative by the reaction of
two creatine molecules and/or by the reaction of a creatine molecule and with a molecule of
creatinine formed from creatine during the derivatization. The peak with shorter retention
time could be due to its TMS ether functionality compared to the keto group.

Molecules 2021, 26, 3206
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◦
Figure 3. NICI (A,C) and PICI (B,D) GC-MS spectra generated from the BSTFA derivatization (60 C, 60 min) of a freshly
prepared solution of d0-creatine in deionized water upon its evaporation to complete dryness. The retention times (tR) of
the GC-MS peak were 14.08 min (minor peak, red) and 14.72 min (major peak, blue). Insets indicate the proposed structures
for the mass fragments. The same oven column temperature program was used as in Figures 1 and 2.
3.3. Standardization of [methylo-2H3]Creatinine
The isotopic purity of stable isotope-labelled analogs is of particular importance in
quantitative analyses [27]. The isotopic purity of the commercially available [methylo-
2H3]creatinine was verified as follows.
Nine separate samples containing each 100 nmol of d0-creatinine and d3-creatinine
were derivatized with BSTFA (100
by SIM of m/z 256, m/z 259, m/z 271, and m/z 274 (peak with retention time 8.9 min).
Analysis of the sample containing d0-creatinine generated a mean PAR of 0.02223 0.00329
(RSD, 15%; n = 9) for m/z 259 to m/z 256, and a PAR of 0.01302 0.00209 (RSD, 16%; n = 9)
for m/z 274 to m/z 271. Analysis of the samples that contained d3-creatinine produced a
mean PAR of 0.000588 0.0001411 (RSD, 24%; n = 9) for m/z 256 to m/z 259 and a mean
PAR of 0.01108 0.0002861 (RSD, 2.6%; n = 9) for m/z 271 to m/z 274. These observations
µL) and 1-µL aliquots of their solutions were analyzed
±
±
±
±
indicate the presence of only very low amounts of d0-creatinine in the commercial [methylo-
2H3]creatinine and confirm its declared isotopic purity (>99 atom% 2H).
3.4. Method Linearity, Precision and Accuracy
For quantitative analyses of creatinine, we selected the N,N,O-tris(trimethylsilyl)
derivative of creatinine with the retention of 8.9 min. The structure of this creatinine
derivative is most likely N2,N2,O4-tris(trimethylsilyl). The structure with the ring-N3 atom
of creatinine, which is not derivatized, allows both PICI and NICI. In the NICI mode, SIM

Molecules 2021, 26, 3206
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of m/z 271 for d0-creatinine and m/z 274 for d3-creatinine was performed. A representative
GC-MS chromatogram is shown in Figure 4 and indicates peaks with closely comparable
intensity (2.66
×
106 versus 2.56
×
106) due to injection of nominally 1 nmol of each analyte.
In the PICI mode, SIM of m/z 256 and m/z 272 for d0-creatinine and of m/z 259 and m/z 275
for d3-creatinine was performed. The dwell-time was 108 ms for all ions and the electron
multiplier voltage was set to 2025 V.
Figure 4. Partial GC-MS chromatograms from the analysis of an equimolar mixture of d0-creatinine (blue) and d3-creatinine
◦
(red) after derivatization with BSTFA at 60 C for 60 min (each 1 nmol injected). SIM of m/z 271 for d0-creatinine and m/z
274 for d3-creatinine was performed in the NICI mode.
Stock solutions (each 20 mM) of d0-creatinine and d3-creatinine were freshly prepared
in Ampuwa deionized water. Dilutions of the stock solution of d0-creatinine were prepared
using Ampuwa water providing d0-creatinine concentrations of 0, 2, 4, 6, 8, 10, 14 and
20 mM. Each 10-µL aliquots of these solutions were combined with each 5-µL aliquots of
the 20 mM d3-creatinine stock solution. After evaporation to dryness under a stream of
nitrogen gas, reconstitution of the residue in absolute ethanol and renewed evaporation to
◦
dryness, derivatization with 100
µ
L BSTFA each was performed (60 min, 60 C). Then. 1-
µL
aliquots of the samples were injected in the splitless mode and analyzed in the PICI mode
by SIM of m/z 256, m/z 259, m/z 272 and m/z 275. The amounts injected were 1 nmol
for d3-creatinine in each sample and varying amounts of d0-creatinine (i.e., 0.0, 0.2, 0.4,
0.6, 0.8, 1.0, 1.4, 2 nmol). These analyses were performed by three persons in triplicate
for each concentration. The precision (relative standard deviation, RSD) ranged between
0.1% and 8.4%. Linear regression analysis between the PAR m/z 256 to m/z 259 (y) or
the PAR m/z 272 to m/z 275 (y) and the amount of d0-creatinine (nmol) (x) for all data
resulted in straight lines with the regression equations y = 0.033 + 0.0087x (r2 = 0.9945) and
y = 0.001 + 0.0098x (r2 = 0.9937), respectively (Figure 5). The reciprocal values of slopes of
the straight lines were 115 nmol and 102 nmol and correspond to the nominal amount
of d3-creatinine of 100 nmol used in the linearity experiment. Thus, SIM of m/z 272 and
m/z 275 yields a higher mean accuracy than SIM of m/z 256 and m/z 259 (87% vs. 98%)
(Figure 5).

Molecules 2021, 26, 3206
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Figure 5. Linear relationships between the peak area ratio (PAR) values and d0-creatinine amounts obtained by SIM of
m/z 256 and m/z 272 for d0-creatinine and of m/z 259 and m/z 275 for d3-creatinine in the PICI mode. The indicated
◦
d0-creatinine amounts and each 100 nmol d3-creatinine were derivatized with BSTFA (100
µ
L) at 60 C for 60 min and 1
µL
aliquots of the reaction mixture were injected in the splitless mode. Data are shown as mean
These analyses were performed by three persons in triplicate for each d0-creatinine amount. For more details, see the text.
±
standard deviation (n = 9).
3.5. Measurement of Creatinine in Human Urine in the NICI Mode
The method was validated in human urine samples in the NICI mode by the three
persons who performed the experiment described above. Three healthy volunteers (#1,
#2, #3) donated urine samples by spontaneous micturition. To 10-µL urine aliquots, d0-
creatinine was added to reach final added concentrations of 2, 4, 6, 10, 14 and 20 mM. d3-
Creatinine was also added to these samples to reach a fixed concentration of 10 mM in each
urine sample. After evaporation to dryness under a stream on nitrogen gas, reconstitution
of the residues in 100
derivatization each with 100
µ
L aliquots of absolute ethanol and renewed evaporation to dryness,
µ
L BSTFA was performed (60 min, 60 ◦C) and 1-
µL aliquots
were injected and analyzed by SIM of m/z 271 and m/z 274 in the NICI mode. Linear
regression analysis between the PAR of m/z 271 to m/z 274 (y) and the concentration of
d0-creatinine (mM) (x) resulted in straight lines (Figure 6). The reciprocal slope values of
the straight lines were 10.9 mM for urine #1, 11.0 mM for urine #2, and 10.2 mM for urine
#3. Based on the nominal concentration of d3-creatinine of 10 mM in the urine samples, the
mean accuracy is calculated to be 109%, 110% and 102% in the three human urine samples
in the concentration range investigated. The y axis intercept values indicate mean basal
creatinine+creatine concentrations of 1.7, 1.8 and 1 mM, respectively.

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Figure 6. Linear relationships between the peak area ratio (PAR) values and the varying d0-creatinine
concentrations added to human urine samples donated by three healthy volunteers and regression
equations. d3-Creatinine was added at the fixed concentration of 10 mM and served as the internal
standard. SIM of m/z 271 and m/z 274 for d0-creatinine and d3-creatinine was performed in the NICI
mode, respectively. The analyses were performed by the three persons who performed the analyses
shown in Figure 5. For more details, see the text.
3.6. HPLC Analysis of Creatinine in HCl Solutions of Creatine
The aim of these analyses was to estimate the extent of formation of creatinine
from creatine and creatine-phosphate in hydrohloric acid solutions of varying molar-
ity and incubation time at room. Linear relationships between the response (y), i.e., peak
area, mAU
y = 7.4 + 4.92 x, (r2 = 0.9999) (range, 0–1000
measure the concentration of creatinine in creatine solutions in hydrochloric acid (Figure 7)
The concentration of creatinine in freshly prepared 5000 M creatine solutions ranged
between 2 and 7 M and increased with increasing HCl molarity and incubation time up
to 56 M at 1 M HCl and 360 min (Figure 7A) and up to 2500–3000 M after 63 days in
250–1000 mM HCl solutions (Figure 7B). The sigmoidal creatinine-incubation time profile
in a 5000 M solution of creatine in 25 mM HCl is shown in (Figure 7C). The highest
creatinine concentration was determined to be 2150 M after 69 days. Creatine-phosphate
×
min at 210 nm, and the creatinine concentration in
µM (x) was observed:
µM). This regression equation was used to
.
µ
µ
µ
µ
µ
µ
was found to be stable in deionized water. Similar experiments with HCl-solutions of
creatine-phosphate did not result in formation of considerable amounts of creatinine (data
not shown). Except for creatine and creatinine we did not detect appearance of additional
peaks within HPLC run time of 5 min and UV absorbance detection at 210, 232 and 250 nm.

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60
50
40
30
20
10
0
A
5 mM Creatine
in:
0 mM HCl
100 mM HCl
250 mM HCl
500 mM HCl
750 mM HCl
1000 mM HCl
0
60
120
180
240
300
360
Incubation time (min)
Figure 7.
(
A
–
C
) Creatinine formed upon incubation of 5 mM creatinine in deionized water and in the indicated HCl
◦
solutions for the indicated times at room temperature (22–25 C). Analyses were performed by HPLC with UV absorbance
detection at 210 nm. Note the double decadic logarithmic scale in panel (C).
4. Discussion
Silylation is one of the most widely used derivatization reaction in analytical chem-
istry, notably in GC-based methods. Silylation reagents such as BSTFA and MSTFA are
not specific, but react with different functionalities of organic compounds, especially of
hydroxyl and amine groups, to form O- and N-trimethylsilyl derivatives [23]. Such deriva-
tives are volatile and thermally stable in non-aqueous systems, best properties in GC-based
analytical methods.
Creatinine, 2-amino-1-methyl-5H-imidazol-4-one (Scheme 1), is an endogenous sub-
stance, the final metabolite of creatine catabolism. Creatinine can be formed chemically
from creatine by acid-catalyzed cyclization (Scheme 1). The most significant field of inter-
est in creatinine is Clinical Chemistry. Serum creatinine serves as an indicator of kidney
function. Urinary creatinine is of particular importance in clinical, pharmacological and
epidemiological studies, where biomarkers must be measured in urine collected from
spontaneous micturition, i.e., when the urine volume and the time between two urine
collections are unknown. This particular importance is because creatinine is excreted in
the urine with a relatively constant rate primarily via glomerular filtration mainly de-
pending on age and gender. The great interest in creatinine in various disciplines led to
the development of many analytical methodologies based on different principles. As an
organic amine, derivatization of creatinine improves its physicochemical properties so
that its analysis becomes feasible by GC also coupled with mass spectrometry (MS) [3–23].
Thus, GC-MS was used several decades ago for the quantitative measurement of creatinine
in biological samples including serum and urine using stable isotope-labelled analogs of
creatinine [20–22].
Using trimethylsilylation (no conditions reported), Lawson found by GC-MS and EI
that creatinine is converted to a single derivative, which was identified as the N,N,O-TMS
derivative [20]. The EI mass spectrum of this derivative contained two ions at m/z 329,

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which is the the molecular radical cation [M]•+, and m/z 314 due to the loss of methyl radical
([M-CH3]+) from one of the three TMS groups [20]. This derivative obviously corresponds
to the derivatives of do-creatinine of d3-creatinine in our study with the retention time of
8.9 min. Siekmann extracted creatinine from human serum samples by cation-exchange
resin Ag 50W-X2, derivatized by N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA)
◦
in anhydrous pyridine (1:1, v/v) by heating (40 min, 60 C) [21]. Siekmann reported on the
formation of a single GC-MS peak (by SIM), of which the EI spectrum was very similar
to that reported by Lawson [20], supporting the formation of a N,N,O-TMS derivative of
creatinine. Neither Lawson nor Siekmann reported in their papers analogous analyses with
creatine.
Our observations strongly suggest that derivatization of creatinine (60 min, 60 ◦C)
with pure BSTFA, i.e., in the absence of any solvents such as pyridine generates at least three
derivatives. The derivative eluting at 8.9 min is most likely N2,N2,O4-tris(trimethylsilyl)-
creatinine, identical with that proposed by Lawson [20] and Siekmann [21]. The second
major derivative formed under the same derivatization conditions is most likely N2,N3,O4-
tris(trimethylsilyl)-creatinine with the retention time of 8.7 min (Scheme 3). This derivative
has not been reported thus far. N2,N3,O4-tris(Trimethylsilyl)-creatinine elutes in front of
N2,N2,O4-tris(trimethylsilyl)-creatinine presumably because all derivatizable N atoms of
creatinine are derivatized.
Scheme 3. Proposed chemical structures for two derivatives of creatinine formed by its reaction with
◦
N,O-tris(trimethylsilyl)trifluoroacetamide (BSTFA) at 60 C for 60 min in pure BSTFA.
Our study also strongly suggests that derivatization of creatine with pure BSTFA under
same conditions (60 min, 60 ◦C) generates the same two derivatives eluting at 8.7 min and
8.9 min. The results from HPLC analyses of creatine solutions in deionized water and
hydrochloric acid solutions suggest that creatine cyclizes to form creatinine, yet a very low
extent. One may therefore assume that the derivatives N2,N3,O4-tris(trimethylsilyl) and
N2,N2,O4-tris(trimethylsilyl) are formed during the BSTFA derivatization step.
To the best of our knowledge, the present study is the first to demonstrate the forma-
tion of two new derivatives from creatine via BSTFA derivatization (60 min, 60 ◦C). These
derivatives emerge from the column 5 to 6 min later than the above-mentioned deriva-
tives of creatinine and creatine. Our study strongly suggests that both late-eluting TMS
derivatives stem from a creatine-creatinine adduct. As no creatinine was initially present

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in the creatine sample, the detected creatine-creatinine is likely to have been formed by
alternative mechanisms. One possible mechanism could involve formation of the O-TMS
ester of creatine (Scheme 4), which is likely to be formed more rapidly and to a higher extent
than the N-TMS [24]. Subsequently, free amine groups may attack the chemically activated
carboxylic group to make the creatinine residues. Thus far, only one group has reported on
the synthesis of creatinyl-amino acids derivatives such as creatinyl-glycine, which has been
reported to be neuroprotective [28]. In the NICI and PICI mass spectra of these derivatives
we obtained mass fragments being each by 4 Da (see Figure 3). An explanation for this
finding could be loss of 4 H atoms in total on the three trimethylsilyl groups of the terminal
guanidine group. We do not know whether this results from the derivatization or ionization
irrespective of the ionization mode. Such a phenomenon has not been reported thus far.
Yet, there is an indication that this may occur in N,N-di-trimethylsilyl derivatives [29–31].
Thus, in the EI mass spectra of the per-trimethylsilylated 1-phosphono-2-amino-ethane
(MW 413) and O-phosphorylethanolamine (MW 429) the cation m/z 174 was observed,
which was assigned to [CH2=N(Si(CH3)3)2]+. These spectra also contained m/z 172 with
intensity ratio of 2:1. A possible structure for m/z 172 could be [CH2=N(Si(CH3)2CH2)2]+.
Scheme 4. Proposed chemical structures for the formation of creatinyl-creatinine derivatives from
◦
the derivatization of creatine with pure N,O-tris(trimethylsilyl)trifluoroacetamide (BSTFA) at 60 C
for 60 min. See the NICI and PICI mass spectra of these derivatives in Figure 3.
Silylation of this compound with a perdeuterated silylation reagent shifted these
cations to m/z 192 and m/z 188 [30], strongly supporting the bridging of two methyl
groups of the neighboring TMS groups on the amine group. BSTFA and other silylation
agents can react with various functionalities [23], including acetamide groups such as
that of acetaminophen (paracetamol) to generate its O,O-di-TMS derivative [31], and their
derivatives undergo multiple fragmentations and rearrangements during ionization such
as EI [32].

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Pentafluoropropionic anhydride (PFPA) is another useful derivatization reagent in
GC-MS. Like BSTFA, PFPA also reacts with amine, hydroxylic and carboxylic groups for
instance of amino acids [33]. The N-pentafluoropropionyl derivatives are considerably
more stable than the O-pentafluoropropionyl derivatives [34]. As BSTFA derivatization
does not allow discrimination between creatinine and creatine, we tested the utility of PFPA.
Under conditions previously re◦ported for amino acids [25
,32], i.e., heating the analytes in
PFPA-ethyl acetate (1:4, v/v; 65 C, 30 min), we observed each only one peak from creatine,
d0-creatinine and d3-creatinine. The GC-NICI-MS spectra of creatine and d0-creatinine
−
−
derivatives were virtually identical: m/z 221 (6 %; [M
−
HF
−
H2O] ), 239 (100 %; [M HF] )
−
−
and m/z 259 (6 %; [M] ; C7H6F5N3O2); the GC-MS spectrum of the d3-creatinine derivative
eluted a few seconds earlier: m/z 224 (6 %), 242 (100 %) and m/z 262 (6 %). These results
indicate the formation presumably of N2-pentafluoropropionyl from both, creatine and
creatinine. These observations suggest that PFPA reacts with the carboxylic group of
creatine to form the mixed anhydride. Subsequently, the N2-imine group attacks intra-
molecularly the carboxylic group, with pentafluoropropionic acid leaving the molecule,
analogous to the BSTFA derivative of creatine.
As far we are informed, creatinine has not be measured by GC-MS in human urine after
derivatization with BSTFA. Our study indicates that creatinine can be quantified precisely
and accurately by GC-MS in only 10-µL aliquots of human urine using d3-creatinine as
internal standard in relevant concentration ranges. The method does not require any
organic solvent or base like pyridine for derivatization and/or extraction for GC-MS
analysis. Excess BSTFA serves as a solvent, in which the TMS derivatives are readily
soluble, yet no other charged endogenous constituents present in urine. As BSTFA is highly
reactive towards numerous substances [23], it is possible that many endogenous substances
also form volatile TMS derivatives that do not accumulate in the GC column.
Currently available data suggest that specific measurement of creatinine in urine
and other biological samples is possible by using 2,3,4,5,6-pentafluorobenzyl (PFB) bro-
mide (PFB-Br) in aqueous acetone (60 min, 50 ◦C) [34]. Creatinine reacts with PFB-Br to
form a single derivative, i.e., N2-PFB-creatinine. Interestingly, we found that the N2-PFB-
◦
derivatives of d0-creatinine and d3-creatinine react with PFPA (65 C, 30 min) to form their
N2-PFB,N3-PFP derivatives (MW=439.21, C14H7F10N3O2; MW=442.23, C14H4D3F10N3O2,
respectively) with relative retention time of 1.29 with respect to the N2-PFB-derivatives.
5. Conclusions
BSTFA is known for many decades as a useful derivatization reagent for the GC-MS
analysis of creatinine, but its utility to measure creatinine in human urine has not been
reported thus far. This study investigated the derivatization of creatinine and its precursor
◦
creatine with BSTFA. Both substances react with BSTFA (60 C, 60 min) to form three
derivatives of v◦irtually identical structures. Creatinine and creatine were found to react
with PFPA (65 C, 30 min) to form a single N-pentafluoropropionyl derivative. These
observations indicate that BSTFA and PFPA is much more effective in the conversion of
creatine to creatinine than lowering the pH by inorganic acids such as hydrochloric acid.
Our findings suggest that BSTFA and PFPA are not useful for the simultaneous measure-
ment of creatinine and creatine. The N,N,O-trimethylsilyl derivative of creatinine and
creatine with the retention time of 8.9 min is useful for their quantitative measurement in
human urine both in the NICI and PICI mode using trideuteromethyl creatinine as internal
standard. Under the same derivatization conditions, creatine reacts with BSTFA and forms
two creatinyl-creatine derivatives with retention times of 14.07 min and 14.72 min, sug-
gesting intermediate formation of creatinine and its conjugation with creatine. Our results
show that methyl groups of the TMS residues react to form -CH2-CH2-bridges and are
supported by previous reports on alkyl amines. Such a cyclization reaction is more likely
to occur during EI, PICI and NICI, rather than during the derivatization with BSTFA. The
possibility that creatinine, but not creatine, reacts with PFB-Br and the N2-PFB-creatinine
derivative reacts with PFPA to form N2-PFB,N3-PFP-creatinine offers the possibility to

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measure biological creatinine and creatine simultaneously by GC-MS. This could be of
particular importance in the area of Clinical Chemistry and in clinical trials.
Author Contributions: Conceptualization, D.T.; methodology, O.B., K.W. and B.B.; software, O.B.
and D.T.; validation, O.B., K.W. and D.T.; formal analysis, D.T.; investigation, O.B., K.W. and B.B.;
resources, D.T.; data curation, O.B., K.W., B.B. and D.T.; writing—original draft preparation, D.T.,
O.B. and K.W.; writing, O.B., K.W. and D.T.; visualization, O.B. and D.T.; supervision, D.T.; project
administration, D.T.; funding acquisition, D.T. All authors have read and agreed to the published
version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Ethical review and approval were waived for this study, due
to the use of spot urine samples donated by three volunteers being authors of this article.
Informed Consent Statement: Patient consent was waived due to the use of spot urine samples
donated by three volunteers being authors of this article.
Data Availability Statement: The study did not report any data.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Not available.
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