A rapid and simple procedure for the determination of synephrine in dietary supplements by gas chromatography-mass spectrometry

A simple and rapid procedure based on gas chromatography-mass spectrometry (GC-MS) is described for determination of synephrine, active principle of Citrus aurantium plant, in solid and liquid dietary supplements. After the addition of 3,4-methylenedioxypropylamphetamine as internal standard (I.S.), a liquid-liquid extraction procedure in alkaline conditions with chloroform/isopropanol (9:1, v/v) was applied to the samples prior to analysis. Chromatography was performed on a fused capillary column and synephrine and I.S., derivatized with pentafluoropropionic anhydride, were determined in the selected-ion-monitoring (SIM) mode. The method was validated in the range 0.1-50 microg/mg or microg/mL synephrine. Mean recovery ranged between 89.3% and 90.5% in both solid and liquid dietary supplements. The quantification limit was 0.1 microg/mg or microg/ml. The method was applied to analysis of various dietary supplements promoted for aiding weight control containing, among other constituents such as ephedrine alkaloids and methylxanthines, Citrus aurantium. Amount of synephrine present in such products ranged from 3.1 microg/mg solid product to 480.2 microg/mL liquid product.     

A rapid and simple procedure for the determination of cannabinoids in hemp food products by gas chromatography-mass spectrometry

A rapid and simple procedure using liquid–liquid extraction and subsequent gas chromatographic mass-spectrometric detection has been developed for determination of Δ⁹-tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) in different hemp foods. After addition of Δ⁸-tetrahydrocannabinol as internal standard, both solid and liquid specimens were extracted with two volumes of 2 ml of hexane/isopropanol (9:1): Chromatography was performed on a fused silica capillary column and analytes were determined in the selected-ion-monitoring (SIM) mode. The method was validated in the range 1–50 ng/ml liquid samples or 1–50 ng/g solid samples for THC and CBN, and 2–50 ng/ml or ng/g for CBD. Mean recoveries ranged between 78.8 and 90.2% for the different analytes in solid and liquid samples. The quantification limits were 1 ng/ml or ng/g for THC and CBN and 2 ng/ml or ng/g CBD. The method was applied to analysis of various hemp foods. THC content in different products varied 50-fold, whereas CBN and CBD were absent in some samples and achieved hundreds of ng/ml or ng/g in others. The concentration ratio (THC + CBN)/CBD was used to differentiate between the phenotypes of cannabis plants in different specimens. Products possibly originating from drug-type cannabis plants were found in the majority of analyzed specimens.     

A simple and reliable procedure for the determination of psychoactive drugs in oral fluid by gas chromatography-mass spectrometry

A simple and reliable gas chromatography-mass spectrometry method for identifying and quantifying psychoactive drugs in oral fluid is described. Substances under investigation were: psychostimulant drugs (amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxiamphetamine, 3,4-methylenedioxy-N-ethylamphetamine, phentermine), cocaine and metabolites (benzoylecgonine, cocaethylene, and ecgonine methyl esther), cannabinoids (delta-9-tetrahydrocannabinol, 11-nor-9-carboxy-delta-9-tetrahydrocannabinol, 11-hydroxy-delta-9-tetrahydrocannabinol, cannabinol and cannabidiol), opiates (6-monoacetylmorphine, morphine and codeine), hypnotics (flurazepam, flunitrazepam, dipotassium chlorazepate, alprazolam, diazepam and oxazepam), antidepressant drugs (amitryptiline, paroxetine and sertraline), antipsychotic drugs (haloperidol, chlorpromazine and fluphenazine) chlormethiazole, loratidine, hydroxyzine, diphenhydramine, valproic acid and gabapentin. After the addition of deuterated analogues of morphine, 3,4-methylenedioxymethamphetamine, (+/-)-11-nor-9-carboxy-delta-9-tetrahydrocannabinol and clonazepam as internal standards, all the compounds were simultaneously extracted from oral fluid by solid-phase extraction procedure. Acid compounds were eluted with acetone while basic and neutral compounds with dichloromethane:isopropanol:ammonium (80:20:2, v/v/v). Chromatography was performed on a methylsilicone capillary column and analytes, derivatized with N-Methyl-N-(trimethylsilyl)trifluoroacetamide, were determined in the selected-ion-monitoring (SIM) mode. Mean recovery ranged between 44.5 and 97.7 % and quantification limit between 0.9 and 44.2 ng/ml oral fluid for the different analytes. The developed analytical methodology was applied to investigate the presence of psychoactive drugs in oral fluid from injured individuals attending the emergency room (MACIUS project).     

Toxic hepatitis in a group of 20 male body-builders taking dietary supplements

Dietary supplements have been used for decades for enhancing muscle growth. The harm caused by some of these products is well documented. We investigated and reported toxic hepatitis in 20 male athletes following self-prescribing of a number of dietary supplements which are lesser known. The patients’ ages ranged from 24 to 32 with a mean of 28 years. They had taken three kinds of supplements for 1 year including testosterone optimizer agent T Bomb II, a creatine supplement Phosphagen and an amino acid based supplement Cell-Tech. Based on the history, clinical examination, and laboratory findings the cases were diagnosed as toxic hepatitis. After discontinuation of taking the supplements, clinical recovery and improvement of liver function tests were achieved within 30 days. Causality assessment with the CIOMS (Council for International Organization Medical Sciences) scale showed a “possible” grade of causality (+5 points) for these supplements. It can be concluded that these newer anabolic supplements may induce toxic hepatitis. Since the health risks of them may be severe, the use of these kinds of dietary supplements should be discouraged.     

A rapid method for chemical fingerprint analysis of Hoodia species, related genera, and dietary supplements using UPLC-UV-MS

Recently, ultra-performance liquid chromatography (UPLC) has proven to be one of the most promising developments in the area of high-speed chromatographic separations with increased sensitivity and resolution. In this work, a reverse phase chromatographic method was developed using UPLC for the chemical fingerprint analysis of 12 hoodigosides, related genera and dietary supplements. The method is also used for the quantification of P57 in Hoodia species and dietary supplements that claim to contain Hoodia. The analysis was performed on a Waters Acquity UPLC system with an Acquity UPLC BEH C18 column (100 mm x 2.1mm I.D., 1.7 microm) and a gradient elution of water and acetonitrile, both containing 0.05% formic acid with a run time of 15 min. The calibration curve of P57 showed good linearity (r(2)>0.999) within the established range (1-100 microg/mL). The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.3 and 0.9 microg/mL, respectively. The RSD for intra- and inter-day were less than 3.0%, and the recovery efficiency as 97-103%. LC-mass spectrometry coupled with electrospray ionization (ESI) interface method is described for the identification of P57. The developed method was successfully applied to the identification of 12 oxypregnane glycosides in four different species of Hoodia, 23 related genera and 35 dietary supplements that claim to contain H. gordonii. The UPLC profiles of various plant samples were compared for the presence of oxypregnane glycosides. Different sample matrices were successfully analyzed, providing the wide range of applicability of this method, including gels, capsules, tablets, sprays, tea bags, snack bars, powders and juices.     

Rapid screening of sulfonamides in dietary supplements based on extracted common ion chromatogram and neutral loss scan by LC-Q/TOF-mass spectrometry

There is an increasing amount of dietary supplements that are adulterated with diuretics and anti-diabetic drugs; this has become a global problem due to the wide distribution of dietary supplements and the serious negative health effects of the adulterants. In this study, a rapid screening method was developed for detection and confirmation of 35 sulfonamides in supplements by ultra-high performance liquid chromatography quadrupole/time of flight mass spectrometry. For effective extraction of sulfonamides from dietary supplements, four extraction protocols including HLB and WAX solid-phase extraction, Quick Easy Cheap Effective Rugged and Safe method, and pH-controlled liquid–liquid extraction were evaluated, and pH-controlled liquid–liquid extraction method was shown to be the most effective with high recovery efficiency and low matrix effect. Rapid separation of 35 sulfonamides was achieved with the UHPLC C18 column (150 × 2.1 mm, 1.7 um) within 7 min using ammonium acetate aqueous solution (pH 8) and acetonitrile as the mobile phase. From the MS/MS spectra of sulfonamides, common ions (m/z 77.9650 [SO₂N]^- and m/z 79.9812 [SO₂NH₂]^-) and neutral molecule loss fragments (HCl and SO₂) were observed according to their structural characteristics. Extracted common ion chromatograms and neutral loss scan of these characteristic fragments could effectively apply for rapid screening of sulfonamides in various types of supplements. A reduced mass tolerance window of ±5 ppm was useful for detecting targeted and untargeted sulfonamides and could avoid false positive and false negative results. Overall calibration curves within dynamic range for all targets were shown to be linear with a correlation coefficient R² > 0.995 and limits of detection ranged from 0.04 to 11.18 ng/g for all sulfonamides. The established method was successfully applied for screening and confirmation of sulfonamides in various supplements. The developed method will be helpful for the identification of sulfonamide diuretics and anti-diabetics in dietary supplements, promoting public health and consumer safety.     

Detection of undeclared erectile dysfunction drugs and analogues in dietary supplements by ion mobility spectrometry

An ion mobility spectrometry (IMS) method was developed to screen for the presence of undeclared synthetic erectile dysfunction (ED) drugs or drug analogues in herbal dietary supplements claiming to enhance male sexual performance. Ion mobility spectra of authenticated reference materials including three FDA approved drugs (sildenafil citrate, tadalafil, vardenafil hydrochloride trihydrate) and five previously identified synthetic analogues (methisosildenafil, homosildenafil, piperidenafil, thiosildenafil, thiomethisosildenafil) were measured to determine their reduced ion mobilities (K₀). All eight compounds exhibited reduced mobilities between 0.8257 and 1.2876 cm²/(V s). Twenty-six herbal products were then screened for the presence of these compounds, and 15 of the 26 products tested positive for the presence of ED drug or drug analogue adulterants based on their reduced ion mobilities. IMS results were compared against the results obtained from an independent LC/MS reference method for the identical samples. Herbal dietary supplements containing adulterants were classified with 100% accuracy and most of the adulterants were correctly identified by a comparison of the K₀ of the adulterant to the K₀ of the authenticated reference material. The results demonstrate that IMS is a viable method for screening herbal dietary supplements for the presence of ED drug or drug analogue adulterants.     

A rapid and simple method for the determination of psychoactive alkaloids by CE‐UV: application to Peganum Harmala seed infusions

The β‐carboline alkaloids of the harmala (HAlks) group are compounds widely spread in many natural sources, but found at relatively high levels in some specific plants like Peganum harmala (Syrian rue ) or Banisteriopsis caapi . HAlks are a reversible Mono Amino Oxidase type A Inhibitor (MAOI) and, as a consequence, these plants or their extracts can be used to produce psychotropic effects when are combined with psychotropic drugs based on amino groups. Since the occurrence and the levels of the HAlks in natural sources are subject to significant variability, more widespread use is not clinical but recreational or ritual, for example B . caapi is a known part of the Ayahuasca ritual mixture. The lack of simple methods to control the variable levels of these compounds in natural sources restricts the possibilities to dose in strict quantities and, as a consequence, limits its use with pharmacological or clinical purposes. In this work, we present a fast, simple, and robust method of quantifying simultaneously the six HAlks more frequently found in plants, i.e., harmine, harmaline, harmol, harmalol, harmane, and norharmane, by capillary electrophoresis instruments equipped with the more common detector UV. The method is applied to analyze these HAlks in P . Harmala seeds infusion which is a frequent intake form for these HAlks. The method is validated in three different instruments in order to evaluate the transferability and to compare the performances between them. In this case, harmaline, harmine, and harmol were found in the infusion samples. Copyright © 2016 John Wiley & Sons, Ltd.     

HPLC法同时测定复方氨茶碱口服液中氨茶碱和盐酸麻黄碱的含量

目的:建立高效液相色谱同时测定复方氨茶碱口服液中氨茶碱和盐酸麻黄碱含量的方法.方法:色谱柱C18柱,流动相为乙腈-磷酸二氢钾缓冲液(pH 4.5)(10:90,V/V),检测波长214 nm,流速0.8 mL/min,检测氨茶碱浓度和盐酸麻黄碱浓度.结果:氨茶碱浓度在20.0～500.0μg/mL,盐酸麻黄碱浓度在2.0～50.0μg/mL范围内线性良好,氨茶碱和盐酸麻黄碱的平均回收率分别为99.7%(RSD=0.58%)、100.1%(RSD=1.31%),n=9.结论:该法简单、灵敏、准确,可用于该复方制剂的质量控制.     

Determination of ephedrine alkaloids in Ephedra natural products using HPLC on a pentafluorophenylpropyl stationary phase

In this study a pentafluorophenylpropyl (PFPP) stationary phase was applied to the fast and reliable qualitative and quantitative analysis of ephedrine alkaloids in Ephedra plant material and derivatives. A Discovery HS F5 column (150mmx4.6mm i.d., 5microm) was used, with an isocratic mobile phase composed of ammonium acetate (7mM) in acetonitrile-water (90:10, v/v), at a flow rate of 1.0ml/min. The column temperature was set at 45 degrees C. UV detection was set at 215 and 225nm. The total analysis time was 16min. The validation parameters, such as linearity, sensitivity, accuracy, precision and specificity, were found to be highly satisfactory. Sonication and microwave extractions were compared in order to optimize the yield of the target analytes. Under the optimized extraction conditions (based on two cycles of sonication with methanol at 40 degrees C for 15min), different matrices containing Ephedra were successfully analyzed for their alkaloid content. The method was applied to the analysis of standard reference materials (SRMs) containing Ephedra. Furthermore, the developed technique allowed the simultaneous determination of ephedrine alkaloids and synephrine, the main phenethylamine alkaloid of Citrus aurantium, that has replaced Ephedra in dietary supplements used in the treatment of obesity. The results indicated that this procedure is suitable for the phytochemical analysis of Ephedra plant material and extracts, and can be applied to demonstrate the label claims for product content, including the absence of ephedrine alkaloids in Ephedra-free products.     

Risk assessment for pyrrolizidine alkaloids detected in (herbal) teas and plant food supplements

Pyrrolizidine alkaloids (PAs) are plant metabolites present in some botanical preparations, with especially 1,2-unsaturated PAs being of concern because they are genotoxic carcinogens. This study presents an overview of tumour data on PAs and points of departure (PODs) derived from them, corroborating that the BMDL₁₀ for lasiocarpine represents a conservative POD for risk assessment. A risk assessment using this BMDL₁₀ and mean levels of PAs reported in literature for (herbal) teas, indicates that consumption of one cup of tea a day would result in MOE values lower than 10 000 for several types of (herbal) teas, indicating a priority for risk management for these products A refined risk assessment using interim relative potency (REP) factors showed that based on the mean PA levels, 7(54%) of 13 types of (herbal) teas and 1 (14%) of 7 types of plant food supplements (PFS) resulted in MOE values lower than 10 000, indicating a priority for risk management also for these products in particular. This includes both preparations containing PA-producing and non-PA-producing plants. Our study provides insight in the current state-of-the art and limitations in the risk assessment of PA-containing food products, especially (herbal) teas and PFS, indicating that PAs in food presents a field of interest for current and future risk management.     

Simultaneous determination of melatonin and pyridoxine in tablets by gas chromatography-mass spectrometry

A gas chromatographic-mass spectrometric (GC–MS) method for the qualitative and quantitative determination of melatonin plus pyridoxine commercial tablets is described. Melatonin and pyridoxine were simultaneously determined by GC–MS after extraction from ground tablets with methanol and derivatization with N-methyl-N-N-trimethlylsilyltrifluoroacetamide (MSTFA). The mass chromatograms were generated using 232 m/z ion for melatonin and 280 m/z ion for pyridoxine, respectively. Splitless injection offers good reproducibility with a standard deviation of 2%. The developed method was applied to analyze the melatonin and pyridoxine content from two different tablet formulations. Also, recovery, detection and quantification limits are reported.     

Causality assessment in hepatotoxicity by drugs and dietary supplements

Structured causality assessment of hepatotoxicity by drugs and dietary supplements (DDS) is a major clinical challenge, since temporal associations as the sole criteria for a valid evaluation are not acceptable. Initially, a clear intuition for an ad hoc evaluation is necessary, but only provisional, and must be followed by a diagnostic algorithm using a pretest, main test and post test. The evaluation is based on a variety of items such as latency period, course of alanine aminotransferase and alkaline phosphatase after DDS discontinuation, risk factors, co-medication, previous information on hepatotoxicity of the DDS, response to rechallenge, and exclusion of other diseases. It is essential that practising and hospital physicians as well as other key health professionals, such as pharmacists, gather all information required for a sound causality assessment, obviating major discussions by expert panels, manufacturers and health agencies in face of scanty and fragmentary data. Because pharmacogenetic alterations may trigger metabolic hepatotoxicity by a few DDS, levels in plasma and urine should be measured and may be helpful for diagnosis. Concomitant genotyping of cytochrome P450 and other enzymes may also be useful in future to minimize the risk of unwanted side-effects, including toxic liver disease elicited by DDS.     

alpha-Lipoic acid in dietary supplements: development and comparison of HPLC-CEAD and HPLC-ESI-MS methods

Alpha-lipoic acid is an antioxidant used both in the prevention and treatment of various oxidative stress related diseases. It is an important constituent of some dietary supplements and can also be found in plant and animal sources. A rapid method for the determination of alpha-lipoic acid in dietary supplements based on high performance liquid chromatography coupled with a coulometric electrode array detector (CEAD) and an electrospray ionization mass spectrometer (ESI-MS) was developed. First, alpha-lipoic acid was extracted with methanol by sonication, chromatographic separation was then achieved by isocratic elution [acetonitrile/methanol/50mM potassium dihydrogen phosphate (pH 3, adjusted with phosphoric acid): 350/65/585, v/v/v] using an ACE 3-C-18 column at a flow rate of 0.45 ml/min. alpha-Lipoic acid was detected by means of a CEAD at +300, +400, +450, +500, +550, +600, +650, and +700 mV against palladium reference electrodes. For ESI-MS detection (negative mode), the composition of the mobile phase was changed to 0.1% acetic acid in water/acetonitrile 55:45, v/v applying a flow rate of 0.2 ml/min. The presented methods were utilized to determine the alpha-lipoic acid content in six dietary supplements. The results of both detection modes were in good correlation.     

Microwave assisted extraction for the determination of ethyl glucuronide in urine by gas chromatography-mass spectrometry

Alcohol is the most frequently abused 'addictive substance' that causes serious social problems throughout the world; thus alcoholism is of particular interest in clinical and forensic medicine. Ethyl glucuronide (EtG) is a marker of recent alcohol consumption that detects alcohol use reliably over a definite time period. The present paper describes a new method for the determination of EtG in urine. It was based both on microwave assisted extraction (MAE) to extract the analyte from urine samples, and gas chromatography-mass spectrometry (GC-MS) to identify and quantify the EtG in selected ion monitoring (SIM) mode. The method was applied to 33 urine samples from alcohol users, obtaining positive results in all cases. It was fully validated including a linear range (0.1-100 microg ml(-1)) and the main precision parameters. In summary, the use of microwave assisted extraction turned out to be a substantially simpler, faster and more sensitive procedure than any other conventional sample preparations.     

Simultaneous determination of amphetamine-type stimulants and cannabinoids in fingernails by gas chromatography-mass spectrometry

A gas chromatography-mass spectrometric (GC-MS) method was developed and validated for the simultaneous detection and quantification of four amphetamine-type stimulants (amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA)) and two cannabinoids (Delta9-tetrahydrocannabinol (Delta9-THC) and 11-nor-Delta9-tetrahydrocannabinol-9-carboxylic acid (THCCOOH)) in fingernails. Fingernail clippings (30 mg) were washed with distilled water and methanol, and then incubated in 1.0 M sodium hydroxide at 95 degrees C for 30 min. The compounds of interest were isolated by liquid-liquid extraction followed by derivatization with N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) at 70 degrees C for 15 min. The derivatized compounds were analyzed by GC-MS in the selective ion monitoring (SIM) mode. The linear ranges were 0.1-15.0 ng/mg for AP, 0.2-15.0 ng/mg for MDA, Delta9-THC and THCCOOH, and 0.2-30.0 ng/mg for MA and MDMA, with good correlation coefficients (r2 > 0.9991). The intra-day, inter-day, and inter-person precisions were within 10.6%, 6.3%, and 5.3%, respectively. The intra-day, inter-day and inter-person accuracies were between -6.1 and 5.0%, -6.2 and 5.7%, and -6.4 and 5.6%, respectively. The limits of detection (LODs) and quantification (LOQs) for each compound were lower than 0.056 and 0.2 ng/mg, respectively. The recoveries were in the range of 74.0-94.8%. Positive GC-MS results were obtained from specimens of nine suspected MA or cannabis abusers. The concentration ranges of MA, AP, and THCCOOH were 0.10-1.41, 0.12-2.64, and 0.20 ng/mg, respectively. Based on these results, the method proved to be effective for the simultaneous qualification and quantification of amphetamine-type stimulants and cannabinoids in fingernails.     

Development and validation of a high-performance liquid chromatography-mass spectrometry assay for methylxanthines and taurine in dietary supplements

A procedure based on liquid chromatography-mass spectrometry (LC-MS) is described for determination of caffeine, theobromine, theophylline, taurine in different dietary supplements. After addition of tryptophan as internal standard, both solid and liquid specimens were extracted with 4 ml of hexane/isopropanol (9:1). Chromatography was performed on a C18 reversed-phase column using water/methanol/acetic acid (75:20:5, v/v/v) as a mobile phase. Analytes were determined in LC-MS single ion monitoring mode with atmospheric pressure ionization-electrospray (ESI) interface. The method was validated in the range 0.1-500 and 0.06-500 microg/ml or microg/g for taurine and caffeine, respectively; 0.06-100 microg/ml or microg/g for theobromine and theophylline. Mean recoveries ranged between 70.1 and 94.4% for different analytes. The quantification limits were 0.1 microg/ml or microg/g for taurine and 0.06 microg/ml or microg/g for methylxanthines either in liquid samples or in solid samples. The method was applied to the analysis of various dietary supplements containing methylxanthines and taurine. Energetic drinks contained amounts of taurine in the range of hundreds to thousands microg/ml and ten times lower amounts of caffeine. Conversely, herbal powders, tablets and capsules mainly contained mg amounts of caffeine per gram of product with the other two methylxanthines in the range of ten to hundred microg/g.     

Simultaneous determination of HFBA-derivatized amphetamines and ketamines in urine by gas chromatography-mass spectrometry

To facilitate the analysis of targeted drugs under high sample volume testing environment, an extraction, derivatization and gas chromatographic-mass spectrometric analysis method was developed for simultaneously determination of amphetamine (AMP), methamphetamine (MAMP), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyethylamphetamine (MDEA), ketamine, and norketamine in urine. This method utilized solid-phase extraction in conjunction with derivatization using heptafluorobutyric anhydride (HFBA) as the derivatization reagent. Using a 1-mL sample, the limits of quantitation achieved for the analysis of AMP, MAMP, MDA, MDMA, MDEA, ketamine, and norketamine were 25, 15, 60, 60, 70, 25, and 30 ng/mL, respectively. Upper limits of quantitation were 8000 ng/mL for all amphetamines and 6000 ng/mL for ketamine and norketamine. Except for dehydronorketamine (DHNK), within-day and between-day precisions (as expressed in CV%) for quality control samples were ≤ 3.1% and ≤ 4.95%, respectively. Except DHNK, the within-day accuracy ranged between 96.0% and 110.7% and the between-day accuracy ranged between 96.9% and 108.7%. A group of 107 urine samples previously determined to contain the target analytes were analyzed by this new approach. Quantitative data produced by both methods agreed well. With this new approach, we were able to use a single analytical protocol to conduct the confirmation test for samples that preliminarily tested positive (by immunoassay) for amphetamines, ketamine, or both.     

Simultaneous determination of spirapril and spiraprilat in plasma by capillary gas chromatography-mass spectrometry

A specific, sensitive and precise method for the simultaneous determination of spirapril (CAS 94841-17-5) and spiraprilat (CAS 83602-05-5) in human plasma is described. The method involves the use of enalapril as internal standard, solid-phase extraction, derivatization and capillary gas chromatography with mass sensitive detection. The working range is from 2.5 to 500 micrograms/l for spirapril and spiraprilat, respectively. Data demonstrating the precision and accuracy of the analytical method are given. Moreover, data concerning freeze-thaw stability, long-term stability of frozen samples, short-term stability of thawed samples, and stability of the extracts in the autosampler are given.     

Simultaneous determination of nitrate and nitrite in human plasma by gas chromatography-mass spectrometry

We devised a sensitive and simple method for the simultaneous determination of nitrate and nitrite in human plasma, using extractive alkylation. These inorganic anions were alkylated with pentafluorobenzyl bromide, using tetradecyldimethylbenzylammonium chloride as the phase-transfer catalyst, with 1,3,5-tribromobenzene as an internal standard. The derivatives were analyzed by gas chromatography-mass spectrometry, using the negative-ion chemical ionization mode with isobutane as the reagent gas. Calibration curves for nitrate and nitrite were linear over the concentration range of 0.01 to 1.0 micromol/mL in plasma, and the lower limit of detection for both compounds was 0.005 micromol/mL. The accuracy and precision of this method were evaluated and coefficients of variation were lower than 10.4%. Blood nitrate and nitrite concentrations of six victims who committed suicide by inhaling automobile exhaust gas could be determined using our method.