Determination of ephedrines in urine by gas chromatography–mass spectrometry

A selective gas–liquid chromatographic method with mass spectrometry (GC–MS) for the simultaneous confirmation and quantification of ephedrine, pseudo-ephedrine, nor-ephedrine, nor-pseudoephedrine, which are pairs of diastereoisomeric sympathomimetic amines, and methyl-ephedrine was developed for doping control analysis in urine samples. O-Trimethylsilylated and N-mono-trifluoroacetylated derivatives of ephedrines — one derivative was formed for each ephedrine — were prepared and analyzed by GC–MS, after alkaline extraction of urine and evaporation of the organic phase, using d₃-ephedrine as internal standard. Calibration curves, with r²>0.98, ranged from 3.0 to 50 μg/ml depending on the analyte. Validation data (specificity, % RSD, accuracy, and recovery) are also presented.     

Quantification of nitrite and nitrate in human urine and plasma as pentafluorobenzyl derivatives by gas chromatography—mass spectrometry using their N-labelled analogs

For the quantification of nitrite and nitrate, the stable metabolites of -arginine-derived nitric oxide (NO) in human urine and plasma, we developed a gas chromatographic—mass spectrometric (GC—MS) method in which [¹⁵N]nitrite and [¹⁵N]nitrate were used as internal standards. Endogenous nitrite and [¹⁵N]nitrite added to acetone-treated plasma and urine samples were converted into their pentafluorobenzyl (PFB) derivatives using PFB bromide as the alkylating agent. For the analysis of endogenous nitrate and [¹⁵N]nitrate they were reduced to nitrite and [¹⁵N]nitrite, respectively, by cadmium in acidified plasma and urine samples prior to PFB alkylation. Reaction products were extracted with toluene and 1-μl aliquots were analyzed by selected-ion monitoring at m/z 46 for endogenous nitrite (nitrate) and m/z 47 for [¹⁵N]nitrite ([¹⁵N]nitrate). The intra- and inter-assay relative standard deviations for the determination of nitrite and nitrate in urine and plasma were below 3.8%. The detection limit of the method was 22 fmol of nitrite. Healthy subjects (n = 12) excreted into urine 0.49 ± 0.25 of nitrite and 109.5 ± 61.7 of nitrate (mean ± S.D., μmol/mmol creatinine) with a mean 24-h output of 5.7 μmol for nitrite and 1226 μmol for nitrate. The concentrations of nitrite and nitrate in the plasma of these volunteers were determined to be (mean ± S.D., μmol/l) 3.6 ± 0.8 and 68 ± 17, respectively.     

Biological monitoring of hexamethylene diisocyanate by determination of 1,6-hexamethylene diamine as the trifluoroethyl chloroformate derivative using capillary gas chromatography with thermoionic and selective-ion monitoring

A GC method using a novel derivatization reagent, 2′,2′,2-trifluoroethyl chloroformate (TFECF), for the derivatization of primary and secondary aliphatic amines with the formation of carbamate esters is presented. The method is based on a derivatization procedure in a two-phase system, where the carbamate ester is formed. The method is applied to the determination of 1,6-hexamethylene diamine (HDA) in aqueous solutions and human urine, using capillary GC. Detection was performed using thermionic specific detection (TSD) and mass spectrometry (MS)—selective-ion monitoring (SIM) using electron-impact (EI) and chemical ionization (CI) with ammonia monitoring both positive (CI)⁺ and negative ions (CI)⁻. Quantitative measurements were made in the chemical ionization mode monitoring both positive and negative ions. Tetra-deuterium-labelled HDA (TDHDA; H₂NC²H₂(CH₂)₄C²H₂NH₂) was used as the internal standard for the GC—MS analysis. In CI⁺ the m/z 386 and the m/z 390 ions corresponding to the [M + 18]⁺ ions (M = molecular ion) of HDA—TFECF and TDHDA—TFECF were measured; in CI⁻ the m/z 267 and the m/z 271 ions corresponding to the [M — 101]⁻ ions. The overall recovery was found to be 97 ± 5% for a HDA concentration of 1000 μg/l in urine. The minimal detectable concentration in urine was found to be less than 20 μg/l using GC—TSD and 0.5 μg/l using GC—SIM. The overall precision for the work-up procedure and GC analysis was ca. 3% (n = 5) for 1000 μg/l HDA-spiked urine, and ca. 4% (n = 5) for 100 μg/l. The precision using GC—SIM for urine samples spiked to a concentration of 5 μg/l was found to be 6.3% (n = 10).     

Determination of nandrolone and metabolites in urine samples from sedentary persons and sportsmen

Metabolites of nandrolone were determined in the urine of several sportsmen, sedentary and post-menopausal women by capillary gas chromatography–mass spectrometry quadrupole (GC–MS) and capillary gas chromatography mass–mass spectrometry ion trap (GC–MS–MS) methods. The method employed was GC–EI-MS with 17α-methyltestosterone as internal standard with ethyl ether extraction prior to selected ion monitoring of the bis(trimethylsilyl) ethers at ion masses m/z 405 and 420 for the nandrolone metabolites, and 418 and 403 for nandrolone derivative. Recovery for nandrolone, 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE) was 97.20, 94.17 and 95.54%, respectively. Detection limits for nandrolone, 19-NA and 19-NE were 0.03, 0.01 and 0.06 ng/ml. Metabolites of nandrolone (19-NA and 19-NE) were found in 12.5% (n=40) of sportsmen and 40% (n=10) of post-menopausal women.     

Simultaneous analysis of the di(2-ethylhexyl)phthalate metabolites 2-ethylhexanoic acid, 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in urine by gas chromatography–mass spectrometry

A gas chromatographic–mass spectrometric method was developed for the quantitative analysis of the three Di(2-ethylhexyl)phthalate (DEHP) metabolites, 2-ethylhexanoic acid, 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in urine. After oximation with O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine hydrochloride and sample clean-up with Chromosorb P filled glass tubes, all three organic acids were converted to their tert.-butyldimethylsilyl derivatives. Quantitation was done with trans-cinnamic acid as internal standard and GC–MS analysis in the selected ion monitoring mode (SIM). Calibration curves for all three acids in the range from 20 to 1000 μg/l showed correlation coefficients from 0.9972 to 0.9986. The relative standard deviation (RSD) values determined in the observed concentration range were between 1.3 and 8.9% for all three acids. Here we report for the first time the identification of 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in human urine next to the known DEHP metabolite 2-ethylhexanoic acid. In 28 urine samples from healthy persons we found all three acids with mean concentrations of 56.1±13.5 μg/l for 2-ethylhexanoic acid, 104.8± 80.6 μg/l for 2-ethyl-3-hydroxyhexanoic acid and 482.2± 389.5 μg/l for 2-ethyl-3-oxohexanoic acid.     

Application of gas chromatography—mass spectrometry with selected ion monitoring to the urinanalysis of 4-pyridoxic acid

An analytical protocol has been developed for the analysis of urinary 4-pyridoxic acid (4-PA) by gas chromatography—mass spectrometry (GC—MS) for use in metabolic studies. Aliquots of urine were deproteinised and fractionated by isocratic reversed-phase high-performance liquid chromatography. The eluent fraction containing the 4-PA was collected, freeze-dried and silylated using N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide. Derivatisation produced the mono-tert.-butyldimethylsilyl derivative of 4-PA lactone. This derivative was readily amenable to GC—MS analysis in the electron ionisation (70 eV) mode, yielding a prominent fragment ion at m/z 222 ([M — 57]⁺; base peak). A heavy isotope-labelled derivative of pyridoxine [dideuteriated pyridoxine; 3-hydroxy-4-(hydroxymethyl)-5-[hydroxymethyl-²H₂]-2-methylpyridine] has been synthesised and is being employed to determine the kinetics of labelling of the body pools of vitamin B₆. Kinetic measurements are based on the determination of the relative proportions of metabolically produced deuterium-labelled and non-labelled 4-PA in urine, obtained from stable isotope ratios determined by low-resolution selected ion monitoring using a bench-top quadrupole GC—MS system.     

Determination of acrolein in human urine by headspace gas chromatography and mass spectrometry

A rapid and sensitive headspace gas chromatographic and mass spectrometric (GC–MS) method was developed for the determination of acrolein in human urine. A 0.5-ml urine sample in a glass vial containing propionaldehyde as an internal standard was heated at 80°C for 5 min. A 0.1-ml volume of headspace vapor was injected into a GC–MS instrument. Acrolein and propionaldehyde were coeluted at 3.1 min using a DB-1 capillary column, and well separated by selective ion monitoring (SIM) mode using ions m/z 56.05 and m/z 58.05. The interassay and intraassay coefficient of variation were 0.99% and 3.3%. The calibration curve demonstrated a good linearity throughout concentrations ranging from 1 to 1000 nM. However, due to a wide variation of acrolein evaporation rates from human urine, a calibration curve must be established for each urine specimen using a standard addition method and detection limit varied from 1 to 5 nM. The total analysis time for two samples from one urine specimen required about 15 min. Therefore, this method is convenient for the urgent monitoring of urinary acrolein in patients to whom alkylating agents are administered.     

GC–MS determination of creatinine in human biological fluids as pentafluorobenzyl derivative in clinical studies and biomonitoring: Inter-laboratory comparison in urine with Jaffé, HPLC and enzymatic assays

In consideration of its relatively constant urinary excretion rate, creatinine in urine is a useful biochemical parameter to correct the urinary excretion rate of endogenous and exogenous biomolecules. Assays based on the reaction of creatinine and picric acid first reported by Jaffé in 1886 still belong to the most frequently used laboratory approaches for creatinine measurement in urine. Further analytical methods for creatinine include HPLC–UV, GC–MS, and LC–MS and LC–MS/MS approaches. In the present article we report on the development, validation and biomedical application of a new GC–MS method for the reliable quantitative determination of creatinine in human urine, plasma and serum. This method is based on the derivatization of creatinine (d₀-Crea) and the internal standard [methyl-trideutero]creatinine (d₃-Crea) with pentafluorobenzyl (PFB) bromide in the biological sample directly or after dilution with phosphate buffered saline, extraction of the reaction products with toluene and quantification in 1-μl aliquots of the toluene extract by selected-ion monitoring of m/z 112 for d₀-Crea-PFB and m/z 115 for d₃-Crea-PFB in the electron-capture negative-ion chemical ionization mode. The limit of detection of the method is 100 amol of creatinine. In an inter-laboratory study on urine samples from 100 healthy subjects, the GC–MS method was used to test the reliability of currently used Jaffé, enzymatic and HPLC assays in clinical and occupational studies. The results of the inter-laboratory study indicate that all three tested methods allow for satisfactory quantification of creatinine in human urine. The GC–MS method is suitable for use as a reference method for urinary creatinine in humans. In serum, creatine was found to contribute to creatinine up to 20% when measured by the present GC–MS method. The application of the GC–MS method can be extended to other biological samples such as saliva.     

Simultaneous determination of eight lipid peroxidation degradation products in urine of rats treated with carbon tetrachloride using gas chromatography with electron-capture detection

One of the major processes that occur as a result of radical-induced oxidative stress is lipid peroxidation (LPO). Degradation of lipid peroxides results in various products, including a variety of carbonyl compounds. In the present study eight different lipid degradation products, i.e., formaldehyde, acetaldehyde, acetone, propanal, butanal, pentanal, hexanal and malondialdehyde were identified and measured simultaneously and quantitatively in rat urine after derivatization with O-(2,3,4,5,6-pentafluorbenzyl)hydroxylamine hydrochloride, extraction with heptane and using gas chromatography–electron-capture detection (GC–ECD). The identity of the respective oximes in urine was confirmed by gas chromatography–negative ion chemical ionization mass spectrometry (GC–NCI-MS). Simultaneously measured standard curves were linear for all oxime-products and the detection limits were between 39.0±5.3 (n=9) and 500±23 (n=9) fmol per μl injected sample. Recoveries of all products from urine or water were 73.0±5.2% and higher. In urine of CCl₄-treated rats an increase in all eight lipid degradation products in urine was found 24 h following exposure. ACON showed the most distinct increase, followed by PROPA, BUTA and MDA. It is concluded that the rapid, selective and sensitive analytical method based on GC–ECD presented here is well suited for routine measurement of eight different lipid degradation products. These products appear to be useful as non-invasive biomarkers for in vivo oxidative stress induced in rats by CCl₄.     

Analysis of tert.-butyldimethylsilyl [1-C]palmitic acid in stool samples by gas chromatography–mass spectrometry with electron impact ionisation: comparison with combustion isotope-ratio mass spectrometry

The use of ¹³C-labelled compounds to study lipid metabolism is increasing. Typically less than 40% of the orally administered label is recovered in breath CO₂. The remainder must be either absorbed and not oxidised or not absorbed and remain in the faeces. Two methods of determining how much tracer passes through the body, and is present in the stool, were compared. Compound specific analysis of tert.-butyldimethylsilyl [¹³C]hexadecanoic acid by gas chromatography–mass spectrometry (GC–MS) with electron impact ionisation was compared with bulk analysis of whole stool and lipid extract by continuous flow isotope ratio mass spectrometry (CF–IRMS) with a combustion interface. The mean difference between the IRMS and GC–MS methods was −0.02 mmol ¹³C d⁻¹ with a mean excretion of 14.2 mmol ¹³C d⁻¹. Combustion IRMS is both simpler and cheaper, when the objective is to determine how much administered dose appears in stool, and information about the form of the label is not required.     

Metabolic profiling of valproic acid by cDNA-expressed human cytochrome P450 enzymes using negative-ion chemical ionization gas chromatography–mass spectrometry

A sensitive negative ion chemical ionization (NCI) gas chromatographic–mass spectrometric (GC–MS) method was modified for the quantitation of valproic acid (VPA) metabolites generated from in vitro cDNA-expressed human microsomal cytochrome P450 incubations. The use of the inherent soft ionization nature of electron-capture NCI to achieve high sensitivity enabled us to conduct kinetic studies using small amounts of recombinant human P450 enzymes. The assay is based on the selective ion monitoring of the intense [M−181] fragments of pentafluorobenzyl (PFB) esters in the NCI mode, and has the following features: (1) a micro-extraction procedure to isolate VPA metabolites from small incubation volumes (100 μl); (2) a second step derivatization with tert.-butyldimethylsilylating reagents to enhance sensitivity for hydroxylated metabolites; (3) a short run-time (<30 min) while maintaining full separation of 15 VPA metabolites by using a narrow-bore non-polar DB-1 column plus a new temperature gradient; and (4) good reproducibility and accuracy (intra- and inter-assay RSDs <15%, bias <15%) by using seven deuterated derivatives of analytes as internal standards. The derivatives of mono- and diunsaturated metabolites, like the parent drug, produced abundant [M−181]⁻ ions while the hydroxylated metabolites gave an ion at m/z of 273, corresponding to the [M−181]⁻ ion of the tert.-butyldimethylsilyl ethers. In conclusion, the GC–NCI-MS analysis of valproate metabolites provided us with a high resolution and sensitivity necessary to conduct metabolic and kinetic studies of valproic acid in small volume samples typical of the in vitro cDNA-expressed micro-incubation enzymatic systems.     

Identification of a flunixin metabolite in camel by gas chromatography–mass spectrometry

A flunixin metabolite, a hydroxylated product, has been identified in camel urine and plasma samples using gas chromatography–mass spectrometry (GC–MS) and GC–MS–MS in the electron impact and chemical ionization modes. Its major fragmentation pattern has been verified by GC–MS–MS in daughter ion and parent ion scan modes. The method could detect flunixin and its metabolite in camel urine after a single intravenous dose of 2.2 mg of flunixin/kg body weight for 96 and 48 h, respectively, which increases the reliability of antidoping control analysis.     

Urinary excretion of 19-norandrosterone of endogenous origin in man: quantitative analysis by gas chromatography–mass spectrometry

A GC–MS method, using deuterium-labelled 19-noretiocholanolone as internal standard and following an extensive LC purification prior to selected ion monitoring of the bis(trimethylsilyl) ethers at ion masses m/z 405, 419, 420 and 421, allowed the quantitation of subnanogram amounts of 19-norandrosterone present in 10-ml urine samples at m/z 405. Thirty healthy men, free of anabolic androgen supply, delivered 24-h urine collections in 4 timed fractions. Accuracy was proven by the equation, relating added (0.05–1 ng/ml) to measured analyte, which had a slope not significantly different from 1. Precision (RSD) was 4% at a concentration of 0.4 ng/ml, and 14% at 0.04 ng/ml. Analytical recovery was 82%. The limit of quantitation was 0.02 ng/ml. The excretion ranges were 0.03–0.25 μg/24 h or 0.01–0.32 ng/ml in nonfractionated 24-h urine.Taking into account inter-individual variability and log-normal distribution, a threshold of 19-norandrosterone endogenous concentration of 2 ng/ml, calculated as the geometric mean plus 4 SD, was established. This value corresponds to the decision limit advised by sport authorities for declaring positive (anabolic) doping with nandrolone.     

Simultaneous determination of imipramine and its metabolite desipramine in human plasma by capillary gas chromatography with mass-selective detection

An analytical method for the simultaneous determination of imipramine (IMI) and its N-desmethyl metabolite, desipramine (DIMI) in human plasma by capillary gas chromatography–mass selective detection (GC–MS), with D4-imipramine (D4-IMI) and D4-desipramine (D4-DIMI) as internal standards, was developed and validated. After addition of the internal standards, the compounds were extracted from plasma at basic pH into n-heptane–isoamyl alcohol (99:1, v/v), back-extracted into acidic aqueous solution and re-extracted at basic pH into toluene. Desipramine and D4-desipramine were converted into their pentafluoropropionyl derivatives. The compounds were determined by gas chromatography using a mass selective detector at m/z 234 for IMI, m/z 238 for D4-IMI, m/z 412 for DIMI and m/z 416 for D4-DIMI. The method was applied to clinical samples.     

Qualitative and quantitative analysis of some synthetic, chemically acting laxatives in urine by gas chromatography—mass spectrometry

A method for the qualitative and quantitative simultaneous analysis of dioxyanthraquinone, desacetyl-Bisacodyl, phenolphthalein and Oxyphenisatin in human urine using gas chromatography—mass spectrometry (GC—MS) has been developed. The compounds were extracted from urine at pH 7.5 with diethyl ether using Extrelut extraction columns, followed by evaporation and trimethylsilylation.The method used electron beam ionization GC—MS employing a computer-controlled multiple-ion detector (mass fragmentography). The recovery from urine for the various compounds was between 80% and 100%. The detection limit for these compounds was in the range 0.01–0.05 μg/ml of urine.The method proved to be suitable for measuring urine concentrations for at least four days after administration of a single oral low therapeutic dose of the laxatives to sixteen healthy volunteers.     

Detection of 4-hydroxycoumarin anticoagulants and their metabolites in urine as part of a systematic toxicological analysis procedure for acidic drugs and poisons by gas chromatography–mass spectrometry after extractive methylation

A gas chromatography–mass spectrometry (GC–MS) procedure was developed for the detection of 4-hydroxycoumarin anticoagulants and their metabolites in urine as part of a systematic toxicological analysis procedure for acidic drugs and poisons after extractive methylation. The part of the phase-transfer catalyst remaining in the organic phase was removed by solid-phase extraction on a diol phase. The compounds were separated by capillary GC and identified by computerized MS in the full scan mode. Using mass chromatography with the ions m/z 291, 294, 295, 309, 313, 322, 324, 336, 343 and 354, the possible presence of 4-hydroxycoumarin anticoagulants and/or their metabolites could be indicated. The identity of positive signals in such mass chromatograms was confirmed by comparison of the peaks underlying full mass spectra with the reference spectra recorded during this study. This method allowed the detection of therapeutic concentrations of phenprocoumon and warfarin in human urine samples. In absence of human urine, acenocoumarol, coumachlor, coumatetrayl, pyranocoumarin (cyclocumarol) could be detected only in rat urine.     

Urine screening of five-day-old newborns: metabolic profiling of neonatal galactosuria

We determined urinary galactose and 4-hydroxyphenyllactic acid (4HPLA) in 4338 of 5-day-old newborns using a newly developed GC–MS screening method. Fifty-two infants were chemically diagnosed as having transient galactosuria based upon elevated urinary galactose levels (4.78–30.53 mg/mg creatinine, control 1.10±0.89 mg/mg creatinine). These infants did not excrete galactitol or galactonic acid into the urine, which is typical of hereditary galactosemia. Nearly 40% of the transient galactosuria was associated with immature infants (low birth weight or borne before 37 gestational weeks). Immature hepatic function is one explanation for neonatal transient galactosuria, but heterozygotes or the carriers of galactose degradation enzyme deficiencies were also suspected in some of the newborns, judging from the comparisons of urinary galactose and 4HPLA excretion between neonates and patients with galactosemia.     

Simplified assay for the quantification of 2,3-dinor-6-ketoprostaglandin F1α by gas chromatography—mass spectrometry

Endogenous prostacyclin production is best assessed by the measurement of its excreted metabolites, of which a major one is 2,3-dinor-6-ketoprostaglandin F_1α (2,3-dinor-6-keto-PGF_1α). Gas chromatographic—mass spectrometric (GC—MS) assays have been developed for this compound but are cumbersome and time-consuming. We now report a modified assay for the measurement of 2,3-dinor-6-keto-PGF_1α employing GC—MS in which sample preparation time is markedly shortened by replacing a number of extraction steps with reversed-phase column extraction and by modifying derivatization procedures. Precision of the assay is ± 5% and the accuracy is 98%. The lower limit of detection in urine is approximately 15 pg/mg creatinine. Normal urinary levels of this metabolite were found to be 141 ± 54 pg/mg creatinine (mean ± S.D.). Urinary excretion of 2,3-dinor-6-keto-PGF_1α is markedly altered in situations associated with abnormalities of prostacyclin generation when quantified using this assay. Thus, this assay provides a sensitive and accurate method to assess endogenous prostacyclin production and to further explore the role of this compound in human health and disease.     

Determination of butorphanol in horse race urine by immunoassay and gas chromatography–mass spectrometry

An analytical procedure to screen butorphanol in horse race urine using ELISA kits and its confirmation by GC–MS is described. Urine samples (5 ml) were subjected to enzymatic hydrolysis and extracted by solid-phase extraction. The residues were then evaporated, derivatized and injected into the GC–MS system. The ELISA test (20 μl of sample) was able to detect butorphanol up to 104 h after the intramuscular administration of 8 mg of Torbugesic, and the GC–MS method detected the drug up to 24 h in FULL SCAN or 31 h in the SIM mode. Validation of the GC–MS method in the SIM mode using nalbuphine as internal standard included linearity studies (10–250 ng/ml), recovery (±100%), intra-assay (4.1–14.9%) and inter-assay (9.3–45.1%) precision, stability (10 days), limit of detection (10 ng/ml) and limit of quantitation (20 ng/ml).     

Gas chromatographic–mass spectrometric identification and quantitation of benzyl alcohol in serum after derivatization with perfluorooctanoyl chloride: a new derivative

Benzyl alcohol is commonly used as an antibacterial agent in a variety of pharmaceutical formulations. Several fatalities in neonates have been linked to benzyl alcohol poisoning. Most methods for measuring benzyl alcohol concentrations in serum utilize direct extraction followed by high-performance liquid chromatography. We describe here a novel derivatization of benzyl alcohol using perfluorooctanoyl chloride after extraction from human serum for analysis by gas chromatography–mass spectrometry (GC–MS). The derivative was eluted at a significantly higher temperature respective to underivatized molecule and the method was free from interferences from more volatile components in serum and hemolyzed specimens. Another advantage of this derivatization technique is the conversion of low-molecular-mass benzyl alcohol (M_r 108) to a high-molecular-mass derivative (M_r 504). The positive identification of benzyl alcohol can be achieved by observing a distinct molecular ion at m/z 504 as well as the base peak at m/z 91. Quantitation of benzyl alcohol in human serum can easily be achieved by using 3,4-dimethylphenol as an internal standard. The within run and between run precisions (using serum standard of benzyl alcohol: 25 mg/l) were 2.7% (mean=24.1, S.D.=0.66 mg/l, n=8) and 4.2% (mean=24.3, S.D.=1.03 mg/l, n=8), respectively. The assay was linear for the serum benzyl alcohol concentrations of 2 mg/l to 200 mg/l and the detection limit was 0.1 mg/l. We observed no carry-over (memory effect) problem in our assay as when 2 μl ethyl acetate was injected into the GC–MS system after analyzing serum specimens containing 200 mg/l of benzyl alcohol, we observed no peak for either benzyl alcohol or the internal standard in the total ion chromatogram.