Stereoselective method development and validation for determination of concentrations of amphetamine-type stimulants and metabolites in human urine using a simultaneous extraction-chiral derivatization approach

Amphetamine-type stimulants (ATS) are a group of chiral amine drugs which are commonly abused for their sympathomimetic and stimulant properties. ATS are extensively metabolised by hepatic cytochrome P450 enzymes. As metabolism of ATS has been shown to be highly stereospecific, stereoselective analytical methods are essential for the quantitative determination of ATS concentrations for both in vivo and in vitro studies of ATS metabolism. This paper describes a new stereoselective method for the simultaneous determination of amphetamine (AM), methamphetamine (MA), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxymethamphetamine (HMMA), 4-hydroxy-3-methoxyamphetamine (HMA), 3,4-hydroxymethamphetamine (HHMA) and 3,4-hydroxyamphetamine (HHA) in human urine samples validated according to the United States Food and Drug Administration guidelines. In this method, analytes are simultaneously extracted and derivatized with R-(-)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (R-MTPCl) as the chiral derivatization reagent. Following this, the analytes were subjected to a second derivatization with N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) which targets the hydroxyl groups present in HMMA, HMA, HHMA and HHA. The derivatized analytes were separated and quantified using gas chromatography-mass spectrometry (GC-MS). The method was evaluated according to the established guidelines for specificity, linearity, precision, accuracy, recovery and stability using a five-day protocol. Intra-day precision ranged from 0.89 to 11.23% RSD whereas inter-day precision was between 1.03 and 12.95% RSD. Accuracy values for the analytes ranged from -5.29% to 13.75%. Limits of quantitation were 10 μg/L for AM, MA, MDMA, HMA and HMMA and 2μg/L for MDA, HMA and HHA. Recoveries and stability values were also within accepted values. The method was applied to authentic ATS-positive samples.     

Simultaneous determination of amphetamine,methamphetamine, 3,4-methylenedioxyamphetamine and 3,4-methylenedioxymethamphetamine in human hair by gas chromatography-mass spectrometry

A procedure is presented for the simultaneous identification and quantification of amphetamine (AP), methamphetamine (MA), methylenedioxyamphetamine (MDA) and methylenedioxymethamphetamine (MDMA) in human hair. The method involves decontamination of hair with dichloromethane and warm water, heat-alkaline hydrolysis in the presence of deuterated internal standards, liquid-liquid extraction and gas chromatography-mass spectrometry after derivatization with pentafluoropropionic anhydride-pentafluoropropanol. The limit of detection for AP, MA and MDA was 0.05 ng/mg using a 50-mg hair sample; for MDMA it was 0.1 ng/mg. Coefficients of variation ranged from 7 to 18%. This assay has been successfully utilized in the evaluation of the deposition of the drugs in hair obtained from various parts of the anatomy of a stimulant abuser.     

Measurement of 7-dehydrocholesterol and cholesterol in hair can be used in the diagnosis of Smith-Lemli-Opitz syndrome

7-dehydrocholesterol (7-DHC) and cholesterol (CHOL) are biomarkers of Smith-Lemli-Opitz Syndrome (SLOS), a congenital autosomal recessive disorder characterized by elevated 7-DHC level in patients. Hair samples have been shown to have great diagnostic and research value, which has long been neglected in the SLOS field. In this study, we sought to investigate the feasibility of using hair for SLOS diagnosis. In the presence of antioxidants (2,6-ditert-butyl-4-methylphenol and triphenylphosphine), hair samples were completely pulverized and extracted by micro-pulverized extraction in alkaline solution or in n-hexane. After microwave-assisted derivatization with N,O-Bis(trimethylsilyl)trifluoroacetamide, the analytes were measured by GC-MS. We found that the limits of determination for 7-DHC and CHOL were 10 ng/mg and 8 ng/mg, respectively. In addition, good linearity was obtained in the range of 50–4000 ng/mg and 30–6000 ng/mg for 7-DHC and CHOL, respectively, which fully meets the requirement for SLOS diagnosis and related research. Finally, by applying the proposed method to real hair samples collected from 14 healthy infants and two suspected SLOS patients, we confirmed the feasibility of hair analysis as a diagnostic tool for SLOS. In conclusion, we present an optimized and validated analytical method for the simultaneous determination of two SLOS biomarkers using human hair.     

Simple and sensitive determination of Delta(9)-tetrahydrocannabinol, cannabidiol and cannabinol in hair by combined silylation, headspace solid phase microextraction and gas chromatography-mass spectrometry

A new method for determination of Delta(9)-tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) in hair based on alkaline hair hydrolysis, extraction by iso-octane, combined derivatization with N,O-bis-(trimethylsilyl)-trifluoroacetamide and headspace solid phase microextraction of the extract residue, and gas chromatography-mass spectrometry was developed and evaluated. The limits of detection of the three compounds were 0.01-0.02 ng/mg. The method was routinely applied to more than 250 hair samples. In 77 positive samples, the concentrations ranged from LOD to 4.2 ng/mg for THC (mean 0.49 ng/mg), to 12.1 ng/mg for CBD (mean 0.37 ng/mg) and to 0.85 ng/mg for CBN (mean 0.12 ng/mg) using a sample amount of 30 mg. The frequently observed increase of the segmental drug concentrations from proximal to distal is explained by progressive accumulation in the hair shaft from sebum or side stream smoke.     

Methodology for the identification of the urinary metabolites of the analgesic-narcotic antagonist, codorphone, by gas chromatography mass spectrometry

Methodology is presented for the identification of codorphone and its metabolites in urine samples using gas chromatography mass spectrometry. The procedure focuses on the clean-up of biological samples and a derivatization technique suitable for these samples. Sep-Pak C-18 cartridges were employed in the clean-up procedure permitting the biological sample to be derivatized in a relatively small volume of reagents. The derivatization procedure incorporated a one-step trimethylsilyloxime reaction to prevent enol formation while simultaneously derivatizing free hydroxyl groups with the excess trimethylsilylimidazole present in the reaction mixture. This was followed by the addition of BSTFA directly to this reaction mixture to complete derivatization of any metabolites possessing dealkylation of the nitrogen. Using this derivatization scheme, synthetic metabolites were analyzed by gas chromatography mass spectrometry, and their mass spectra were characterized emphasizing the diagnostic fragment ions observed in the spectra. To illustrate the usefulness of this methodology, a urine sample obtained from a dog that had been dosed with codorphone was analyzed by gas chromatography mass spectrometry, and the metabolites were identified by comparison to the mass spectra of the synthetic derivatives.     

Validation of a simultaneous analytical method for the detection of 27 benzodiazepines and metabolites and zolpidem in hair using LC-MS/MS and its application to human and rat hair

Benzodiazepines and zolpidem are controlled in many countries due to their inherent adverse effects of a high degree of tolerance and dependence. Recently, as some of these drugs have become distributed illegally and available through media such as the Internet, their abuse is becoming a serious social problem. Hair is a useful specimen to prove chronic drug use. In the present study, a simultaneous analytical method for the detection of 27 benzodiazepines and metabolites and zolpidem in hair was established and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The drugs and their metabolites in hair were extracted using methanol, filtered and injected on the LC-MS/MS. The following validation parameters of the method were satisfactory: selectivity, linearity, matrix effect, recovery, process efficiency, intra- and inter-assay precision and accuracy and processed sample stability. The limit of detection (LOD) and the limit of quantification (LOQ) were the total drug detected from the sample. The LODs ranged from 0.005 ng (zolpidem) to 0.5 ng (bromazepam and chlordiazepoxide) and the LOQs were 0.25 ng in every analyte except for bromazepam and chlordiazepoxide, for which they were 0.5 ng. The developed method was successfully applied to five legal cases involving use of benzodiazepines and zolpidem and to an animal study on drug incorporation into hair. Diazepam and its three metabolites, as well as lorazepam, were detected in hair from both the multiple- and single-dose administration groups of lean Zucker rats. The concentration of diazepam was higher than those of its metabolites in both dark grey and white hair from the multiple-dose administration groups, with the mean concentration ranges from 0.16 to 0.51 ng/mg and from 0.10 to 0.24 ng/mg, respectively. The mean concentration ranges of lorazepam were from 0.05 to 0.37 ng/mg in dark grey hair and from 0.11 to 0.45 ng/mg in white hair from the multiple-dose administration groups. Hair pigmentation did not have any significant effect on the degree of the deposition of drugs and their metabolites in hair.     

Stable isotope-coded quaternization for comparative quantification of estrogen metabolites by high-performance liquid chromatography-electrospray ionization mass spectrometry

A fast and sensitive LC-ESI-MS method is described for the comparative quantification of 16 estrogen metabolites based on the derivatization of estrogens with a novel derivatizing reagent, N-methyl-nicotinic acid N-hydroxysuccinimide ester (C1-NA-NHS). The process introduces a quaternary amine to the analytes, making the analytes permanently charged regardless of the pH of the high-performance liquid chromatography (HPLC) mobile phase. This quaternization resulted in a highly efficient separation of 16 estrogen metabolites in 7 min at a detection level below 1 ng/mL. By using a deuterated derivatizing reagent (C1-d(3)-NA-NHS), a complete set of deuterated standards was utilized and used as internal standards in a comparative quantification and recovery study, demonstrating acceptable results over a wide concentration range. A pooled breast cancer serum sample was analyzed using the described method, and 15 estrogens were detected in the range of 80-530 pg/mL.     

p-Toluenesulfonyl isocyanate as a novel derivatization reagent to enhance the electrospray ionization and its application in the determination of two stereo isomers of 3-hydroxyl-7-methyl-norethynodrel in plasma

In this paper, p-toluenesulfonyl isocyanate has been reported as a novel derivatization reagent with strong nuclephilic reactivity for the hydroxyl compounds. The derivatization for the two pharmacologically active 3-hydroxyl metabolites, 3alpha-hydroxyl-7-methyl-norethynodrel and 3beta-hydroxyl-7-methyl-norethynodrel by p-toluenesulfonyl isocyanate can be accomplished in 2 min under room temperature. The offline derivatization procedure introduced an easily ionizable sulfonylcarbamic ester moiety to the metabolites. This greatly improved the analyte's sensitivity in negative electrospray ionization and enabled us to achieve the desired lower limit of quantitation at 100 pg/ml in plasma. Therefore, a sensitive high performance liquid chromatography-mass spectrometry (HPLC-MS) method for the analysis of the two stereo isomers was developed. The method had been validated to be accurate, precise, and sensitive, and can be used for the metabolism pharmacokinetic study of tibolone in human subjects.     

A method for concurrent diazomethane synthesis and substrate methylation in a 96-sample format

In the emerging field of metabolomics, there is an increasing need for improving sample derivatization reactions for gas chromatographic-mass spectral analysis of metabolites with large numbers of samples. This protocol details the safe direct derivatization of organic acids using diazomethane in a 96-sample format. Diazomethane is a highly reactive gas that readily forms methyl esters with carboxylic functionalities, with minimal side products or nonvolatile reaction residues. However, diazomethane's reactivity and explosive potential make it hazardous to store and work with. In this procedure, diazomethane is generated in situ and used concurrently to methylate up to 96 samples simultaneously, thus reducing concerns about reagent stability and obviating the need for storage of solutions of the highly reactive gas. Once the diazomethane generator has been assembled, processing 96 samples takes 2-3 h using this procedure.     

Simultaneous assay of cocaine, heroin and metabolites in hair, plasma, saliva and urine by gas chromatography—mass spectrometry

As part of an ongoing research program on the development of drug detection methodology, we developed an assay for the simultaneous measurement of cocaine, heroin and metabolites in plasma, saliva, urine and hair by solid-phase extraction (SPE) and gas chromatography—mass spectrometry (GC—MS). The analytes that could be measured by this assay were the following: anhydroecgonine methyl ester; ecgonine methyl ester; ecgonine ethyl ester; cocaine; cocaethylene; benzoylecgonine; cocaethylene; norcocaethylene; benzoylnorecgonine; codeine; morphine; norcodeine; 6-acetylmorphine; normorphine; and heroin. Liquid specimens were diluted, filtered and then extracted by SPE. Additional handling steps were necessary for the analysis of hair samples. An initial wash procedure was utilized to remove surface contaminants. Washed hair samples were extracted with methanol overnight at 40°C. Both wash and extract fractions were collected, evaporated and purified by SPE. All extracts were evaporated, derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) and analyzed by GC—MS. The limit of detection (LOD) for cocaine, heroin and metabolites in biological specimens was approximately 1 ng/ml with the exception of norcodeine, normorphine and benzoylnorecgonine (LOD = 5 ng/ml). The LOD for cocaine, heroin and metabolites in hair was approximately 0.1 ng/mg of hair with the exception of norcodeine (LOD = 0.3 ng/mg) and normorphine and benzoylnorecgonine (LOD = 0.5 ng/mg). Coefficients of variation ranged from 3 to 26.5% in the hair assay. This assay has been successfully utilized in research on the disposition of cocaine, heroin and metabolites in hair, plasma, saliva and urine and in treatment studies.     

Determination of triazolam involving its hydroxy metabolites in hair shaft and hair root by reversed-phase liquid chromatography with electrospray ionization mass spectrometry and application to human hair analysis

A sensitive method using reversed-phase liquid chromatography coupled with electrospray ionization mass spectrometry has been developed for simultaneous determination of triazolam and its hydroxy metabolites in hair. After the addition of deuterium-labeled 1-hydroxymethyltriazolam as an internal standard, the analytes in hair shaft and hair root samples were extracted with a basic medium, CH(2)Cl(2):MeOH:28% NH(4)OH (20:80:2) at room temperature overnight. The chromatographic separation of the analytes was achieved using a semimicro HPLC column (3-microm particle size; 100 x 2.0-mm i.d.) by gradient elution with acetonitrile in water containing 1% acetic acid as eluent. The mass spectrometer was operated in selected-ion monitoring mode at quasi-molecular ions [M+H](+) of triazolam and its metabolites. The method has been applied to determine the incorporation of triazolam and its metabolites into the hair shafts and hair roots of Dark Agouti rats administered 3 or 6 mg/kg triazolam intraperitoneally twice a day for 5 days. Triazolam, 1-hydroxymethyltriazolam, and 4-hydroxytriazolam were incorporated into the hair shafts and the hair roots. The concentration of 4-hydroxytriazolam was the highest of all compounds detected. An unknown substance considered to be 1,4-dihydroxytriazolam also appeared in the hair samples. The structural elucidation was performed with online HPLC-MS after acetylation of the substance with acetic anhydride and pyridine. The time course studies of triazolam and the metabolites in both rat hair roots and plasma were carried out after single intraperitoneal administration of triazolam. The concentrations of triazolam and the metabolites in the hair roots reflected those in the plasma. The proposed method using selected-reaction monitoring was applied to the determination of triazolam and the metabolites in human hairs of a triazolam addict. Triazolam, 1-hydroxymethyltriazolam, and 4-hydroxytriazolam were identified in the black hair shafts, whereas only triazolam was detected in the hair roots and the white hair shafts. This is the first report on the detection of triazolam and its metabolites in human hairs.     

On-line coupling of sequential injection with liquid chromatography for the automated derivatization and determination of gamma-aminobutyric acid in human biological fluids

The principle of sequential injection analysis (SIA) was exploited to develop a rapid fully automated and efficient pre-column derivatization procedure coupled on-line to liquid chromatography (HPLC). Using the SIA-HPLC derivatization protocol gamma-aminobutyric acid (GABA) was determined fluorimetrically in human biological fluids with o-phthaldialdehyde (OPA) as derivatization reagent and minimum sample pretreatment. A lab-built SIA system was used to handle samples, standard solutions and OPA reagent. Appropriate volumes of the reagents were introduced in the holding coil of the SIA system and were mixed on propulsion to the HPLC loop through a suitable reaction coil. The chemical (pH, c(OPA), c(mercaptoethanol)) and instrumental variables (volumes of sample and reagent, reaction time) of the reaction were studied and optimized in terms of maximum sensitivity. The chromatographic variables (gradient composition of the eluent and flow rate) were studied for optimum selectivity and peak characteristics. The developed experimental configuration facilitated fully-automated operation thus minimizing errors in handling. Additionally the method as a whole provided very satisfactory sensitivity, precision and accuracy. Direct determination of GABA in human urine and cerebrospinal fluid (CSF) at microg L(-1) (ppb) levels was accomplished, with minimum sample pretreatment.     

Mass spectral characterization of doxylamine and its rhesus monkey urinary metabolites

This study describes the use of mass spectrometry (MS), high-performance liquid chromatography (HPLC) and chemical derivatization techniques for the identification of doxylamine and five rhesus monkey urinary metabolites. The analyses were performed using chemical ionization mass spectrometry with either methane or ammonia as the reagent gas. The confirmation of the structures of two of these urinary metabolites was aided by the synthesis of doxylamine N-oxide and desmethyldoxylamine and by the use of methylation and acetylation derivatization techniques. Doxylamine N-oxide, desmethyldoxylamine, didesmethyldoxylamine, and two metabolites which resulted from the cleavage of the aliphatic tertiary nitrogen side chain to the subsequent 2-[1-phenyl-1-(2-pyridinyl)ethoxy]acetic acid or 2-[1-phenyl-1-(2-pyridinyl)ethoxy]methanol compounds were isolated and identified from rhesus monkey urine. Additional data concerning the mass spectral analysis of derivatization or reaction products from the three chloroformate reactions with doxylamine, and the synthesis and separation techniques which afforded mass spectral identification of the urinary metabolites are also presented.     

Micellar electrokinetic chromatography separation and laser-induced fluorescence detection of the lipid peroxidation product 4-hydroxynonenal

4-Hydroxnonenal (HNE) is a product of lipid peroxidation in biological systems that causes a variety of harmful biological effects. A method for identifying HNE based on derivatization with the fluorescent reagent dansylhydrazine (5-(dimethylamino)naphthalene-1-sulphonehydrazine (DNSH) followed by micellar electrokinetic chromatography separation laser-induced fluorescence detection has been developed. The derivatization reaction has also been investigated for significant experimental parameters and rat brain homogenates with induced lipid peroxidation have been analysed for HNE contents. The limit of detection (3 S/N) was 30 nM or 0.3 fmol in the injected sample.     

Rapid screening procedure based on headspace solid-phase microextraction and gas chromatography-mass spectrometry for the detection of many recreational drugs in hair

An increasing number of synthetic drugs are appearing on the illicit market and on the scene of drug use by youngsters. Official figures are underestimated. In addition, immunochemical tests are blind to many of these drugs and appropriate analytical procedures for routine clinical and epidemiological purposes are lacking. Therefore, the perceived increasing abuse of recreational drugs has not been proved yet. In a previous paper, we proposed a procedure for the preliminary screening of several recreational substances in hair and other biological matrices. Unfortunately, this procedure cannot apply to cocaine. Consequently, we performed a new headspace solid-phase microextraction and gas chromatography-mass spectrometry (HS-SPME-GC-MS) procedure for the simultaneous detection of cocaine, amphetamine (A), methamphetamine (MA), methylen-dioxyamphetamine (MDA), methylen-dioxymethamphetamine (MDMA), methylen-dioxyethamphetamine (MDE), N-methyl-1-(1,3-benzodioxol-5-yl)-2-butanamine (MBDB), ketamine, and methadone in human hair. Hair was washed with water and acetone in an ultrasonic bath. A short acid extraction with 1M hydrochloric acid was needed; the fiber was exposed to a 5 min absorption at 90 degrees C and thermal desorption was performed at 250 degrees C for 3 min. The procedure was simple, rapid, required small quantities of sample and no derivatization. Good linearity was obtained over the 0.1-20.0 ng/mg range for the target compounds. Sensitivity was good enough: limits of detection (LOD) were 0.7 ng/mg of hair for the majority of substances. The intra-day precision ranged between 7 and 20%. This paper deals with the analytical performance of this procedure and its preliminary application to hair samples obtained on a voluntary basis from 183 young people (138 males and 45 females) in the Rome area.     

Analysis of cocaine and three of its metabolites in hair by gas chromatography-mass spectrometry using ion-trap detection for CI/MS/MS

A sensitive GC/CI/MS/MS method was developed for the simultaneous determination of cocaine (COC), anhydroecgonine methylester (cocaine pyrolysis product, AEME), ecgonine methylester (cocaine enzymatic hydrolysis product, EME) and cocaethylene (cocaine with ethanol trans-esterification product, COET) in human hair samples. After acid hydrolysis, hair samples were extracted with an automated solid phase extraction (SPE). The analysis of cocaine and its three metabolites was performed using an ion-trap spectrometer in positive chemical ionization with isobutane as gas reagent. The procedure was validated. Weighted linear regression was found appropriate in a concentration range of 0.10-5.00 ng/mg for AEME, 0.05-5.00 ng/mg for COC, EME and COET. The limit of detection was estimated at 0.005 ng/mg for COC and COET, at 0.025 ng/mg for EME, and at 0.050 ng/mg for AEME. Method performance was evaluated in terms of trueness and precision using quality control (QC) samples over the investigated ranges. Method selectivity and robustness were also demonstrated.     

Sensitive determination of pheomelanin after 5-carboxyfluorescein succinimidyl ester precapillary derivatization and micellar electrokinetic capillary chromatography with laser-induced fluorescence detection

The suitability of micellar electrokinetic capillary chromatography (MEKC) with laser-induced fluorescence (LIF) detection for the determination of pheomelanin in biological materials has been investigated. 5-Carboxyfluorescein succinimidyl ester was chosen as the labeling reagent to precapillary derivatize the two marker aminohydroxyphenylalanine (AHP) isomers produced after reductive hydrolysis of pheomelanin with hydriodic acid (HI). Various parameters affecting derivatization and separation were systematically studied. Under optimal conditions, the analytes could be separated within 18 min, and the relative standard deviations (R.S.D.) of migration time and corrected peak areas were less than 5.5%. Compared with the conventional high-performance liquid chromatography (HPLC) method with electrochemical detection, the 100-fold improvements in sensitivity were achieved by applying LIF detection. As a preliminary application, this method has been successfully applied to the determination of pheomelanin in two human melanoma cell cultures, black hair, melanoma tissue and urine samples of human melanoma patients with the spiked recoveries in the range of 88-96%.     

Analysis of dialkyl phosphate metabolites in hair using gas chromatography-mass spectrometry: A biomarker of chronic exposure to organophosphate pesticides

The aim of our study was to develop and validate an analytical approach for the quantitative determination of three dialkyl phosphate (DAP) metabolites, dimethyl phosphate (DMP), dimethyl thiophosphate (DMTP) and diethyl phosphate (DEP), of organophosphate pesticides (OPs) in hair samples. The proposed methodology comprises a decontamination step, solid–liquid extraction, followed by liquid–liquid extraction, pentafluorobenzyl bromide derivatization, clean-up on Florisil/PSA column and analysis by gas chromatography–mass spectrometry (GC-MS). Extraction recovery, obtained from 50?mg hair samples spiked at two concentration levels, ranged from 56.1 to 107.9% and the within-day precision ranged from 13.5 to 17.5%. Limits of detection (LODs) ranged from 0.02 to 0.10?ng mg^?1. The results obtained from the analysis of hair samples of 30 agricultural workers show the suitability of the proposed method for monitoring people occupationally exposed to OPs. The most frequently detected compound was DEP followed by DMP. This is the first report on the detection of dialkyl phosphates in human hair which reflects the ability of hair testing to assess chronic exposure to OPs.     

Derivatization of carboxylic acids with 4-APEBA for detection by positive-ion LC-ESI-MS(/MS) applied for the analysis of prostanoids and NSAID in urine

In order to develop a generic positive ionization ESI LC-MS method for a variety of interesting substance classes, a new derivatization strategy for carboxylic acids was developed. The carboxylic acid group is labeled with the bromine containing 4-APEBA reagent based on carbodiimide chemistry. The derivatization reaction can be carried out under aqueous conditions, thereby greatly simplifying sample preparation. In this paper, the derivatization of carboxylic acids is exemplified for the determination of prostanoids and non-steroidal anti-inflammatory drugs (NSAID). Optimization of the derivatization conditions was studied. In order to prove the applicability of the presented approach, we applied the described protocol to urine samples from complex regional pain syndrome (CRPS) patients and were able to detect several prostanoids not visible in the urine of healthy volunteers. Further, the determination of the non-steroidal anti-inflammatory drug ibuprofen in a urine sample was possible.