Liquid chromatography-electrospray mass spectrometry determination of carbamazepine, oxcarbazepine and eight of their metabolites in human plasma

Carbamazepine (CBZ) and oxcarbazepine (OXCBZ) are both antiepileptic drugs, which are prescribed as first-line drugs for the treatment of partial and generalized tonic-clonic epileptic seizures. In this paper, a specific and sensitive liquid chromatography-electrospray ionization mass spectrometry method was described for the simultaneous determination of carbamazepine (CBZ), oxcarbazepine (OXCBZ) and eight of their metabolites [CBZ-10,11-epoxide (CBZ-EP), 10,11-dihydro-10,11-trans-dihydroxy-carbamazepine (DiOH-CBZ), 10-hydroxy-10,11-dihydroCBZ (10-OH-CBZ), 2-hydroxycarbamazepine (2-OH-CBZ), 3-hydroxycarbamazepine (3-OH-CBZ), iminostilbene (IM), acridone (AO) and acridine (AI)] in human plasma. The work-up procedure involved a simple precipitation with acetone. Separation of the analytes was achieved within 50 min using a Zorbax eclipse XD8 C8 analytical column. The mobile phase consisted of a mixture of acetonitrile-formate buffer (2 mM, pH 3). Detection was performed using a quadrupole mass spectrometer fitted with an electrospray ion source. Mass spectrometric data were acquired in single ion recording mode at m/z 237 for CBZ, m/z 180 for CBZ-EP and AI, m/z 236 for OXCBZ, m/z 237 for 10-OH-CBZ, m/z 253 for 2-OH-CBZ, 3-OH-CBZ and DiOH-CBZ, m/z 196 for AO and m/z 194 for IM. For all analytes, the drug/internal standard peak height ratios were linked via a quadratic relationship to plasma concentrations. The extraction recovery averaged 90% for CBZ, 80% for OXCBZ and was 80-105% for the metabolites. The lower limit of quantitation was 0.5mg/l for CBZ, 0.4 mg/l for OXCBZ and ranged from 0.02 to 0.3 mg/l for the metabolites. Precision ranged from 2 to 13% and accuracy was between 86 and 112%. This method was found suitable for the analysis of plasma samples collected during therapeutic drug monitoring of patients treated with CBZ or OXCBZ.     

Liquid chromatography-electrospray ionization ion trap mass spectrometry for analysis of mesocarb and its metabolites in human urine

A method is described for the determination of metabolites of mesocarb in human urine by combining gradient liquid chromatography and electrospray ionization (ESI)-ion trap mass spectrometry. Seven metabolites (two isomers of hydroxymesocarb, p-hydroxymesocarb, two isomers of dihydroxymesocarb and two isomers of trihydroxymesocarb) and parent drug were detected in human urine after the administration of a single oral dose 10 mg of mesocarb (Sydnocarb, two tablets of 5 mg). Various extraction techniques (free fraction, enzyme hydrolyses and acid hydrolyses) and their comparison were carried out for investigation of the metabolism of mesocarb. After extraction procedure the residue was dissolved in methanol and injected into the column HPLC (Zorbax SB-C18 (Narrow-Bore 2.1 x 150 mm i.d., 5 microm particles)) with mobile phase (0.2 ml/min) of methanol/0.2 mM ammonium acetate. Conformation of the results and identification of all metabolites are performed by LC-MS and LC-MS/MS. The major metabolites of mesocarb in urine of the human were p-hydroxylated derivative of the phenylcarbamoyl group of the parent drug (p-hydrohymesocarb) and dihydroxylated derivative of mesocarb (two isomers of dihydroxymesocarb). This analytical method for dihydrohymesocarb was very sensitive for discriminating the ingestion of mesocarb longer than the parent drug or other metabolites in human urine. The dihydroxymesocarb was detected in urine until 168-192 h after administration of the drug.     

Liquid chromatography-electrospray mass spectrometry determination of free and total concentrations of ropivacaine in human plasma

A specific and sensitive liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method was developed for the determination of free and total ropivacaine in human plasma. The work-up procedure involved a simple precipitation of plasma proteins with methanol. Etidocaine served as the internal standard. After microscale equilibrium-dialysis, measurement of free ropivacaine levels was performed after direct injection of the dialysate into the chromatograph. The system used a Zorbax eclipse XD8 C8 analytical column packed with 5 microm diameter particles as the stationary phase. The mobile phase consisted of a 15-min gradient (mobile phase A: 0.05% (v/v) trimethylamine in acetonitrile, mobile phase B: 2mM ammonium formate buffer (pH 3)). Mass spectrometric data were acquired in single ion monitoring mode at m/z 275 for ropivacaine and m/z 277 for etidocaine. The drug/internal standard peak area ratios (plasma) or peak areas (dialysate) were linked via a quadratic relationship to concentrations. Precision ranged from 1 to 7.6% accuracy was between 92.6 and 109%. The lower limits of quantitation were 1 microg/l in plasma and 2 microg/l in the dialysate. This method was found suitable for the analysis of plasma samples collected during a clinical trial performed in 30 infants undergoing epidural anaesthesia or continuous psoas compartment block.     

Determination of dansylated monoamine and amino acid neurotransmitters and their metabolites in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry

The determination of neurotransmitters (NTs) and their metabolites facilitates better understanding of complex neurobiology in the central nervous system disorders and has expanding uses in many other fields. We present a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) method for the quantification of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), norepinephrine (NE), vanillymandelic acid (VMA), 3-methoxy-4-hydroxy phenylglycol (MHPG), 5-hydroxytryptamine (5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA), glutamate (Glu), and γ-aminobutyric acid (GABA). The NTs and their metabolites were dansylated and analyzed by an LC gradient on a C18 column on-line with a tandem mass spectrometer. This method exhibited excellent linearity for all of the analytes with regression coefficients higher than 0.99. The lower limit of quantification (LLOQ) values for DA, DOPAC, HVA, NE, VMA, MHPG, 5-HT, 5-HIAA, Glu, and GABA were 0.57, 0.37, 0.35, 0.40, 0.35, 0.91, 0.27, 0.43, 0.65, and 1.62 pmol/ml, respectively. The precision results were expressed as coefficients of variation (CVs), ranging from 1.5% to 13.6% for intraassay and from 2.9% to 13.7% for the interassay. This novel LC-ESI/MS/MS approach is precise, highly sensitive, specific, and sufficiently simple. It can provide an alternative method for the quantification of the NTs and their metabolites in human plasma.     

Low-level quantitation of oxycodone and its oxidative metabolites, noroxycodone, and oxymorphone, in rat plasma by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry

A method was developed for quantification of oxycodone, noroxycodone, and oxymorphone in small volumes (50 microl) of rat plasma by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry using turbo ion-spray. Deuterated (d3) opioid analogues acted as internal standards. Sample preparation involved protein precipitation with acetonitrile, centrifugal evaporation, and reconstitution in mobile phase; analyte separation was performed on a C18 (5 microm, 2.1 mm x 50 mm) column using a linear gradient program. Lower limits of quantitation (ng/ml) and their between-day accuracy and precision were-oxycodone, 0.9 (-0.2 and 7.8%); noroxycodone, 1.0 (0.6 and 6.2%); oxymorphone 1.0 (-1.8 and 9.5%).     

Liquid chromatography-electrospray ionisation mass spectrometry for the determination of nine selected benzodiazepines in human plasma and oral fluid

A new simple and rapid liquid chromatographic-mass spectrometric technique was designed for the determination of nine benzodiazepines in plasma and oral fluid. Benzodiazepines were extracted from alkalinised spiked and clinical plasma and oral fluid samples using a single step, liquid-liquid extraction procedure with diethyl ether. The chromatographic separation was performed with a Xterra RP18, 5 microm (150 x 2.1 mm i.d.) reversed-phase column using deuterated analogues of the analytes as internal standard. The recovery ranged from 70.3 to 86.9% for plasma and 63.9 to 77.2% for oral fluid. The limits of detection ranged from 0.5 to 1 ng/ml in plasma and 0.1 to 0.2 ng/ml for oral fluid. The method was validated for all the compounds, including linearity and the main precision parameters. The procedure, showed to be sensitive and specific, was applied to real plasma and oral fluid samples. The method is especially useful to analyse saliva samples from drivers undergoing roadside drug controls.     

High performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-MS/ESI) method for simultaneous determination of venlafaxine and its three metabolites in human plasma

A high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-MS/ESI) method for simultaneous determination of venlafaxine (VEN) and its three metabolites O-desmethylvenlafaxine (ODV), N-desmethylvenlafaxine (NDV) and N,O-didesmethylvenlafaxine (DDV) in human plasma has been developed and validated. Estazolam was used as the internal standard. The compounds and internal standard were extracted from plasma by a liquid-liquid extraction. The HPLC separation of the analytes was performed on a Thermo BDS HYPERSIL C18 (250 mm x 4.6 mm, 5 microm, USA) column, using a gradient elution program with solvents constituted of water (ammonium acetate: 30 mmol/l, formic acid 2.6 mmol/l and trifluoroacetic acid 0.13 mmol/l) and acetonitrile (60:40, V/V) at a flow-rate of 1.0 ml/min. All of the analytes were eluted within 6 min. The compounds were ionized in the electrospray ionization (ESI) ion source of the mass spectrometer and were detected in the selected ion recording (SIR) mode. Calibration curves in spiked whole blood were linear from 4.0-700 ng/ml, 2.0-900 ng/ml, 3.0-800 ng/ml and 2.0-700 ng/ml for VEN, ODV, NDV and DDV, respectively, all of them with coefficients of determination above 0.9991. The average extraction recoveries for all the four analytes were above 77%. The methodology recoveries were higher than 91%. The limits of detection were 0.4, 0.2, 0.3, and 0.2 ng/ml for VEN, ODV, NDV and DDV, respectively. The intra- and inter-day variation coefficients were less than 11%. The method is accurate, sensitive and reliable for the pharmacokinetic study of venlafaxine as well as therapeutic drug monitoring (TDM).     

Use of high performance liquid chromatography-electrospray ionization-tandem mass spectrometry for the analysis of ceramide-1-phosphate levels

Ceramide-1-phosphate (C1P) is a bioactive sphingolipid with roles in several biological processes. Currently, high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC ESI-MS/MS) offers the most efficient method of quantifying C1P. However, the published protocols have several drawbacks causing overestimations and carryovers. Here, the reported overestimation of C1P was shown to be due to incomplete neutralization of base hydrolyzed lipid extracts leading to the hydrolysis of SM to C1P. Actual quantity of C1P in cells (6 pmols/10⁶ cells) was much lower than previously reported. Also, the major species of C1P produced by ceramide kinase (CERK) was found to be d_18:1/16:0 with a minority of d_18:1/24:1 and d_18:1/24:0. The artifactual production of C1P from SM was used for generating C1Ps as retention time markers. Elimination of carryovers between samples and a 2-fold enhancement in the signal strength was achieved by heating the chromatographic column to 60^°C. The role of ceramide transport protein (CERT) in supplying substrate to CERK was also revalidated using this new assay. Finally, our results demonstrate the presence of additional pathway(s) for generation of the C1P subspecies, d_18:1/18:0 C1P, as well as a significant portion of d_18:1/16:0, d_18:1/24:1, and d_18:1/24:0. In conclusion, this study introduces a much improved and validated method for detection of C1P by mass spectrometry and demonstrates specific changes in the C1P subspecies profiles upon downregulation of CERK and CERT.     

Liquid chromatography-electrospray mass spectrometry of tunicamycin-type antibiotics

Several Streptomyces and Clavibacter species produce a family of tunicamycin-like antibiotics (tunicamycins, streptovirudins, corynetoxins, etc.) that inhibit the polyprenol-P:N-acetylhexosamine-1-P translocase family, thus blocking both bacterial cell wall biosynthesis and eukaryotic protein N-glycosylation. The mechanisms of biosynthesis and resistance to these toxins by the producing bacteria are largely unknown. Electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) mass spectrometric techniques are described that structurally assign tunicamycin N-acylated variants in the picomolar range. A voltage gradient across the ESI inlet port was used to generate fragmentation ions that were structurally diagnostic for the tunicamycins. The application of in-line reversed-phase high-performance liquid chromatography-electrospray MS (LC-ESI-MS) resulted in the identification of eight new tunicamycins. Based on these structural assignments a revised nomenclature for tunicamycins is proposed. Application of the LC-ESI-MS methodology to culture supernatants and cellular extracts of the tunicamycin-producing bacterium, Streptomyces lysosuperificus, confirmed tunicamycin production and showed it to be growth-temperature dependent, but did not detect corynetoxins production in culture by phage-infected Clavibacter toxicus.     

Analysis of testosterone and dihydrotestosterone in mouse tissues by liquid chromatography-electrospray ionization-tandem mass spectrometry

A novel method was established for simultaneous quantitation of testosterone (T) and dihydrotestosterone (DHT) in murine tissue and serum samples. Endogenous T and DHT, together with the internal standards 17α-methyl-T and 17α-methyl-DHT, were extracted from tissues and then derivatized by reaction with 2-hydrazino-4-(trifluoromethyl)-pyrimidine (HTP). Analysis by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) resulted in product ion spectra of HTP derivatives of both T and DHT that showed analyte-specific fragmentations; the latter fragmentations were characterized by the use of high-resolution Orbitrap MS/MS. These specific fragmentations enabled quantitation of T and DHT in the multiple-reaction monitoring (MRM) mode. The method was validated with charcoal-stripped serum as the matrix. The lower limit of quantitation (LLOQ) was 0.10 ng/ml for T and 0.50 ng/ml for DHT. The method was then used for determination of serum and tissue levels of T and DHT in transgenic mice carrying a hypomorphic NADPH-cytochrome P450 reductase gene (Cpr-low mice). Remarkably, ovarian T levels in Cpr-low mice were found to be 25-fold higher than those in wild-type mice, a finding that at least partly explains the female infertility seen in the Cpr-low mice. In conclusion, our method provides excellent sensitivity and selectivity for determination of endogenous levels of T and DHT in mouse tissues.     

High-throughput and simultaneous measurement of homocysteine and cysteine in human plasma and urine by liquid chromatography-electrospray tandem mass spectrometry

Total homocysteine (tHcy) and cysteine (tCys) concentrations in biological fluids are routinely used in the clinical diagnosis of genetic and metabolic diseases, and this necessitates the development of rapid and sensitive methods for quantification. Liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) was used to measure tHcy and tCys in 23 plasma and 21 urine samples from healthy adults and 14 urine samples from healthy children. The results were compared with a standard high-performance liquid chromatography (HPLC) method. The coefficient of variation (CV) for the LC-MS/MS method ranged from 2.9% to 6.1% for the intraassay and 4.8% to 6.4% for the interassay. Mean recoveries were close to 100% for both plasma and urinary tHcy and tCys. The mean plasma tHcy and tCys concentrations in healthy adults were 8.62 and 261.40 micromol/L, respectively. The mean urinary tHcy and tCys in adults were 0.98 and 22.60 micromol/mmol creatinine, respectively. The mean urinary tHcy and tCys in children were 1.17 and 27.43 micromol/mmol creatinine, respectively. Bland-Altman difference plots of method comparison between LC-MS/MS and HPLC showed good agreement in plasma and urinary tHcy and tCys concentrations. Our method is suitable for rapid measurements, and the reported urinary values in children will help to develop a pediatric reference range for clinical use.     

Determination of carotenoids and all-trans-retinol in fish eggs by liquid chromatography-electrospray ionization-tandem mass spectrometry

A novel method was developed for the combined determination of carotenoids and retinoids in fish eggs, which incorporates prior analyte isolation using liquid-liquid partitioning to minimize analyte degradation, and fraction analysis using high-performance liquid chromatography-electrospray (positive)-quadrupole mass spectrometry (LC-ESI(+)-MS; SIM or MRM modes). Eggs from Chinook salmon (Oncorhynchus tshawytscha) were used as the model fish egg matrix. The methodology was assessed and validated for beta-carotene, lutein, zeaxanthin, and beta-cryptoxanthin (molecular ion radicals [M](+)), canthaxanthin and astaxanthin ([M+Na](+) adducts) and all-trans-retinol ([(M+H)-H(2)O](+)). Using replicate egg samples (n=5) spiked with beta-cryptoxanthin and beta-carotene before and after extraction, matrix-sourced ESI(+) enhancement was observed as evidenced by comparable %matrix effect and %process efficiency values for beta-cryptoxanthin and beta-carotene of 114-119%. In aquaculture-raised eggs from adult Chinook salmon astaxanthin, all-trans-retinol, lutein and canthaxanthin were identified and determined at concentrations of 4.12, 1.06, 0.12 and 0.45 microg/g (egg wet weight), respectively. To our knowledge, this is the first report on a method for LC-MS determination of carotenoids and retinoids in a fish egg matrix, and the first carotenoid-specific determination in any fish egg sample.     

Lansoprazole quantification in human plasma by liquid chromatography-electrospray tandem mass spectrometry

An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection was developed for the determination of lansoprazole in human plasma using omeprazole as the internal standard. The analyte and internal standard were extracted from the plasma samples by liquid-liquid extraction using diethyl-ether-dichloromethane (70:30; v/v) and chromatographed on a C(18) analytical column. The mobile phase consisted of acetonitrile-water (90:10; v/v)+10 mM formic acid. The method has a chromatographic total run time of 5 min and was linear within the range 2.5-2000 ng/ml. Detection was carried out on a Micromass triple quadrupole tandem mass spectrometer by Multiple Reaction Monitoring (MRM). The intra- and inter-run precision, calculated from quality control (QC) samples, was less than 3.4%. The accuracy as determined from QC samples was less than 9%. The method herein described was employed in a bioequivalence study of two capsule formulations of lansoprazole.     

Lisinopril quantification in human plasma by liquid chromatography-electrospray tandem mass spectrometry

An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection was developed for the determination of Lisinopril in human plasma using Enalaprilat as internal standard. The analyte and internal standard were extracted from the plasma samples by solid-phase extraction using Waters HLB Oasis SPE cartridges and chromatographed on a C8 analytical column. The mobile phase consisted of acetonitrile/water (60:40, v/v) + 20 mM acetic acid + 4.3 mM of triethylamine. The method had a chromatographic total run-time of 6.5 min and was linear within the range 2.00-200 ng/ml. Detection was carried out on a Micromass triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM). The precision (CV%) and accuracy, calculated from limit of quantification (LOQ) samples (n = 8), were 8.9 and 98.9%, respectively. The method herein described was employed in a bioequivalence study of two tablet formulations of Lisinopril 20mg.     

Liquid chromatography tandem mass spectrometry method for simultaneous determination of metoprolol tartrate and ramipril in human plasma

A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of metoprolol tartrate (MT) and ramipril, in human plasma. Both the drugs were extracted by liquid-liquid extraction with diethyl ether-dichloromethane (70:30, v/v). The chromatographic separation was performed on a reversed-phase C8 column with a mobile phase of 10 mM ammonium formate-methanol (3:97, v/v). The protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The method was validated over the concentration range of 5-500 ng/ml for metoprolol and ramipril in human plasma. The precursor to product ion transitions of m/z 268.0-103.10 and m/z 417.20-117.20 were used to measure metoprolol and ramipril, respectively.     

Survey of albumin purification methods for the analysis of albumin-organic toxicant adducts by liquid chromatography-electrospray ionization-tandem mass spectrometry

HSA has been shown to react with many organic toxicants to form adducts that are useful biomarkers for exposure. Albumin isolation is an important first step for the analysis of these protein-toxicant adducts. We tested several approaches to isolate albumin from serum treated with an electrophilic organic toxicant known to form adducts with albumin, i.e., sulfur mustard agent (HD) (2,2'-dichloroethyl sulfide), in order to evaluate these techniques as purification methods. To select the most efficient isolation strategy, methods were evaluated using gel electrophoresis, total protein quantitation, and peptide-adduct identification by MS. Results suggest that the albumin-rich fractions obtained can be used to identify exposure by quantitating the albumin adducts to electrophilic organic toxicants such as HD. The HiTrap Blue HP albumin isolation system appears to display the most promising results for purifying albumin to detect HD-adducts, exhibiting high purification efficiency, satisfactory albumin recovery, promising specificity, and a higher loading capacity for serum.     

Quantitation of tamsulosin in human plasma by liquid chromatography-electrospray ionization mass spectrometry

A highly sensitive method for quantitation of tamsulosin in human plasma using 1-(2,6-dimethyl-3-hydroxylphenoxy)-2-(3,4-methoxyphenylethylamino)-propane hydrochloride as the internal standard (I.S.) was established using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). After alkalization with saturated sodium bicarbonate, plasma were extracted by ethyl acetate and separated by HPLC on a C18 reversed-phase column using a mobile phase of methanol-water-acetic acid-triethylamine (620:380:1.5:1.5, v/v). Analytes were quantitated using positive electrospray ionization in a quadrupole spectrometer. LC-ESI-MS was performed in the selected ion monitoring (SIM) mode using target ions at m/z 228 for tamsulosin and m/z 222 for the I.S. Calibration curves, which were linear over the range 0.2-30 ng/ml, were analyzed contemporaneously with each batch of samples, along with low (0.5 ng/ml), medium (3 ng/ml) and high (30 ng/ml) quality control samples. The intra- and inter-assay variability ranged from 2.14 to 8.87% for the low, medium and high quality control samples. The extraction recovery of tamsulosin from plasma was in the range of 84.2-94.5%. The method has been used successfully to study tamsulosin pharmacokinetics in adult humans.     

Sensitive determination of vinorelbine and its metabolites in human serum using liquid chromatography-electrospray mass spectrometry

A liquid chromatography-electrospray mass spectrometry method was developed for the quantitation of vinorelbine (VNB) and two metabolites, vinorelbine N-oxide (VNO) and deacetyl vinorelbine (DAV) in human serum. The limits of quantitation (LOQ) reached 0.5 ng/ml for both VNB and VNO and 1 ng/ml for DAV. The method was proved linear in the range of LOQs up to 1000 ng/ml, and extraction recovery was 80% on average for the three compounds. It was applied to the pharmacokinetic monitoring of vinorelbine and, for the first time, to the detection of VNO in the serum of patients suffering from non-small-cell lung cancer.     

Comparison of rapid liquid chromatography-electrospray ionization-tandem mass spectrometry methods for determination of glycoalkaloids in transgenic field-grown potatoes

Two rapid methods for highly selective detection and quantification of the two major glycoalkaloids in potatoes, alpha-chaconine and alpha-solanine, were compared for robustness in high-throughput operations for over 1000 analytical runs using potato tuber samples from field trials. Glycoalkaloids were analyzed using liquid chromatography coupled to tandem mass spectrometry in multiple reaction monitoring mode. An electrospray interface was used in the detection of glycoalkaloids in positive ion mode. Classical reversed phase (RP) and hydrophilic interaction (HILIC) columns were investigated for chromatographic separation, ruggedness, recovery, precision, and accuracy. During the validation procedure both methods proved to be precise and accurate enough in relation to the high degree of endogenous biological variability found for field-grown potato tubers. However, the RP method was found to be more precise, more accurate, and, more importantly, more rugged than the HILIC method for maintaining the analytes' peak shape symmetry in high-throughput operation. When applied to the comparison of six classically bred potato cultivars to six genetically modified (GM) lines engineered to synthesize health beneficial inulins, the glycoalkaloid content in potato peels of all GM lines was found within the range of the six cultivars. We suggest complementing current unbiased metabolomic strategies by validating quantitative analytical methods for important target analytes such as the toxic glycoalkaloids in potato plants.     

The quantitative analysis of heroin, methadone and their metabolites and the simultaneous detection of cocaine, acetylcodeine and their metabolites in human plasma by high-performance liquid chromatography coupled with tandem mass spectrometry

For a pharmacokinetic-pharmacodynamic study in opioid tolerant patients, who were treated with heroin in combination with methadone, a liquid chromatographic assay with tandem mass spectrometry detection (LC-MS/MS) was developed for the simultaneous determination of heroin, methadone, heroin metabolites 6-monoacetylmorphine, morphine, and morphine-6 and 3-glucuronide and methadone metabolite EMDP. To detect any abuse of substances besides the prescribed opioids the assay was extended with the detection of cocaine, its metabolites benzoylecgonine and norcocaine and illicit heroin adulterants acetylcodeine and codeine. Heroin-d6, morphine-d3, morphine-3-glucuronide-d3 and methadone-d9 were used as internal standards. The sample pre-treatment consisted of solid phase extraction using mixed mode sorbent columns (MCX Oasis). Chromatographic separation was performed at 25 degrees C on a reversed phase Zorbax column with a gradient mobile phase consisting of ammonium formate (pH 4.0) and acetonitrile. The run time was 15 min. MS with relatively mild electrospray ionisation under atmospheric pressure was applied. The triple quadrupole MS was operating in the positive ion mode and multiple reaction monitoring (MRM) was used for drug quantification. The method was validated over a concentration range of 5-500 ng/mL for all analytes. The total recovery of heroin varied between 86 and 96% and of the heroin metabolites between 76 and 101%. Intra-assay and inter-assay accuracy and precision of all analytes were always within the designated limits (< or =20% at lower limit of quantification (LLQ) and < or =15% for other samples). This specific and sensitive assay was successfully applied in pharmacokinetic studies with medically prescribed heroin and toxicological cases.