Chromatography-electrospray Ionization-tandem Mass Spectrometry Method Research Articles

Quantitative analysis of nine coumarins in rat urine and bile after oral administration of Radix Glehniae extract by high‐performance liquid chromatography–electrospray ionization tandem mass spectrometry

Coumarins are the primary bioactive ingredients in Radix Glehniae, named Beishashen in China, which possesses many pharmacological activities, including anticancer, anti-inflammation and antivirus activities. In the present study, we employed a sensitive and selective high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method for the quantification of nine coumarins in rat urine and bile: scopoletin (1), xanthotoxol (2), xanthotoxin (3), psoralen (4), isoimpinellin (5), bergapten (6), oxypeucedanin (7), imperatorin (8) and isoimperatorin (9). Pimpinellin (10) was used as the internal standard (IS). The urine and bile samples were pretreated by liquid-liquid extraction with ethyl acetate (EtOAc). The chromatographic separation was carried out on a C₁₈ column with gradient elution. The detection of analytes was performed on a tandem mass system equipped with a turbo ion spray interface in positive mode using multiple-reaction monitoring (MRM). The specificity, linearity, accuracy, precision, recovery, matrix effect and several stabilities were validated for coumarins in rat urine and bile samples. The results showed that this method is robust, specific and sensitive and it can successfully fulfill the requirements of the excretion study of the nine coumarins in Radix Glehniae.

Determination of endocrine‐disrupting compounds in drinking waters by fast liquid chromatography–tandem mass spectrometry

Growing attention has been recently paid to safety of food and drinking water, making necessary the adoption of policies for water sources protection and the development of sensitive and rapid analytical methods to identify micropollutants. Endocrine-disrupting compounds (EDCs) have emerged as a major issue as they alter the functioning of the endocrine system. Since ingestion of EDCs via food is considered the major exposure route, there is a growing interest in understanding EDC fate during drinking water treatment and in monitoring potential contamination of surface waters and groundwaters. In this work, a fast liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed for the determination of 4-n-nonylphenol (NP), bisphenol A (BPA), estrone (E1), 17β-estradiol (E2) and 17α-ethinylestradiol (EE2) in drinking waters. In the literature analytical articles seldom provide details regarding fragmentation pathways. In this paper spectra of the five EDCs in negative ESI were interpreted with the support of accurate mass spectra acquired by a quadrupole time-of-flight instrument; fragmentation pathways were also proposed. The chromatographic separation of EDCs was optimized on a Pinnacle DB Biphenylic column with a water-acetonitrile gradient. Quantitative analysis was performed in multiple reaction monitoring (MRM) mode using bisphenol A-d(16) (BPA-d(16)) as internal standard; calibration curves showed good correlation coefficients (0.9989-0.9997). All figures of merit of the method were satisfactory; limits of detection were in the range 0.2-0.4 ng/ml. The method was applied to the determination of the analytes in waters sampled by polar organic chemical integrative samplers in a drinking water treatment plant. Rather low concentration of BPA, NP and E1 were measured in the inlet, while none of the considered EDCs was detected in the outlet.

Trace detection of the chlorohydrins of epoxidized soybean oil in foodstuffs by UPLC–ESI–MS/MS

Epoxidized soybean oil (ESBO) is used as an authorized plasticizer and a stabilizer for plastic polymers such as poly(vinyl chloride) (PVC). Recently, however, there has been a concrete effort devoted to its substitution for other plasticizers such as polyadipates. ESBO is exploited particularly in food closure gaskets for metal lids used to seal glass jars and bottles. The closure gaskets form an airtight seal necessary to prevent microbiological contamination. Thus, there are potential uses for food sterilization and storage. Additionally, the main pathway of PVC degradation involves the elimination of HCl, which can react with the epoxy groups of ESBO to give mono-, polychlorohydrins and/or other cyclic derivatives. The European Food Safety Authority noted that not enough analytical and toxicological data exist to express a formal opinion on the significance for the health effects of such derivatives. At present in the scientific literature, there are only a few indicative results of direct measurements of ESBO derivatives and there are no official analytical methods available for the determination of chlorohydrins directly from foodstuffs. This study presents the first example of the analysis of commercial food sauces for the detection of ESBO-chlorohydrins (as methyl esters). The results are obtained by a dedicated development of an ultraperformance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method. Sample preparation was based on the following main steps: organic extraction, transesterification and solid-phase extraction clean up. In particular, four isomers for 18-E-OHCl chlorohydrin and eight isomers for 18-2OHCl chlorohydrin were separated and identified. Different food sauces samples closed in glass jars with twist-off caps were subjected to qualitative determination, which yielded positive results for 18-E-OHCl, whereas no traces of 18-2OHCl were found.

Development of a fast liquid chromatography–tandem mass spectrometry method for the determination of endocrine-disrupting compounds in waters

A fast liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) method was developed to study five endocrine-disrupting compounds (4-n-nonylphenol, bisphenol A, estrone, 17β-estradiol and 17α-ethinylestradiol) in water. Different columns were tested; the chromatographic separation of the analytes was optimized on a Pinnacle DB biphenylic column with a water-acetonitrile gradient elution, which allowed the separation of the selected endocrine-disrupting compounds (EDCs) in less than 6 min. Quantitative analysis was performed in selected reaction monitoring (SRM) mode; two transitions were chosen for each compound, using the most abundant for quantitation. Calibration curves using bisphenol A-d (16) as internal standard were drawn, showing good correlation coefficients (0.9993-0.9998). All figures of merit of the method were satisfactory; limits of detection were in the low pg range for all analytes. The method was then applied to the determination of the analytes in real water samples: to this aim, polar organic chemical integrative samplers (POCIS) were deployed in the influent and in the effluent of a drinking water treatment plant in Liguria (Italy). The EDC level was rather low in the influent and negligible in the outlet, reflecting the expected function of the treatment plant.

Determination of free and glucuronidated kaempferol in rat plasma by LC–MS/MS: Application to pharmacokinetic study

Flavanoid kaempferol is mainly present as glucuronides and sulfates in rat plasma, and small amounts of the intact aglycone are also detected. In the this study, a rapid, specific and sensitive liquid chromatography–electrospray ionization-tandem mass spectrometry method (HPLC–MS/MS) was developed and validated for determination of kaempferol and its major metabolite glucuronidated kaempferol in rat plasma. A liquid–liquid extraction with acetic ether was involved for the extraction of kaempferol and internal standard. Analytes were separated on a C18 column (150 mm × 2.1 mm, 4.5 μm, Waters Corp.) with isocratic elution at a flow-rate of 0.3 ml min −1. The mobile phase was consisted of 0.5% formic acid and acetonitrile (50:50, v/v). The Quattro Premier HPLC–MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. The method was validated according to the FDA guidelines for validation of bioanalytical method. The validated method was successfully applied to the study of the pharmacokinetics in rats after oral administration of kaempferol with different doses.

Abstract 1689: Liquid chromatography-electrospray ionization-tandem mass spectrometry analysis for 7-carboxymethylguanine and 7-carboxyethylguanine in human liver DNA

Abstract The carcinogenic nitrosamines N-nitrososarcosine (NSAR) and 3-(methylnitrosamino)propionic acid (MNPA) have been detected in the diet, tobacco products, and human urine. Metabolic activation of NSAR by methyl hydroxylation would produce a DNA carboxymethylating agent while similar metabolism of MNPA would produce a carboxyethylating agent, each of which would be expected to react at the 7 position of guanine to give 7-carboxymethylguanine (7-CMG) and 7-carboxyethylguanine (7-CEG), respectively. Furthermore, acrylic acid, a major industrial product, environmental contaminant and likely endogenous metabolite, and β-propiolactone, are also known to react with DNA to give 7-CEG. We developed a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS-SRM) method to quantify these adducts in human liver DNA. [15N5]7-CMG and [15N5]7-CEG were synthesized and used as internal standards. After addition of the internal standards, the DNA was hydrolyzed enzymatically and the hydrolysate was partially purified by solid-phase extraction. The fraction containing 7-CMG and 7-CEG was treated with acetyl chloride in methanol to convert them to the corresponding methyl esters. After re-purification by solid-phase extraction, the appropriate fraction was analyzed by LC-ESI-MS/MS-SRM for the transitions m/z 224[M + H]+ → m/z 164[(M + H) - C2H4O2]+ for 7-CMG and m/z 238[M + H]+ → m/z 152 [BH]+ for 7-CEG. Human liver DNA samples had clear peaks co-eluting with [15N5]7-CEG, but no peaks were observed at the retention time of 7-CMG. The on column detection limits were approximately 10 and 5 fmol, respectively, for the methyl esters of 7-CMG and 7-CEG. The detection limit for the methyl ester of 7-CEG in DNA (starting with 1 mg) was approximately 8 fmol. Known addition studies using 7-CEG and calf thymus DNA demonstrated that the method was accurate and precise. Artifactual formation of 7-CEG during DNA isolation and enzyme hydrolysis was excluded. The method was applied for the analysis of 27 human liver samples. 7-CEG, but not 7-CMG, was detected in each sample, mean ± S.D, 408 ± 375 fmol/µmol guanine (82 adducts per 109 nucleotides), range 17 − 1491 fmol/µmol guanine. This is the first report demonstrating the presence of 7-CEG in DNA from humans. While MNPA, β-propiolactone and acrylic acid are possible sources of this DNA adduct, we consider acrylic acid to be the most likely because of its ubiquitous occurrence and probable endogenous formation from the lipid peroxidation product acrolein. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1689.

Determination of cymipristone in human plasma by liquid chromatography–electrospray ionization-tandem mass spectrometry

A rapid, specific and sensitive liquid chromatography–electrospray ionization-tandem mass spectrometry method was developed and validated for determination of cymipristone in human plasma. Mifepristone was used as the internal standard (IS). Plasma samples were deproteinized using methanol. The compounds were separated on a ZORBAX SB C 18 column (50 mm × 2.1 mm i.d., dp 1.8 μm) with gradient elution at a flow-rate of 0.3 ml/min. The mobile phase consisted of 10 mM ammonium acetate and acetonitrile. The detection was performed on a triple-quadruple tandem mass spectrometer by selective reaction monitoring (SRM) mode via electrospray ionization. Target ions were monitored at [M+H] + m/ z 498 → 416 and 430 → 372 in positive electrospray ionization (ESI) mode for cymipristone and IS, respectively. Linearity was established for the range of concentrations 0.5–100 ng/ml with a coefficient correlation ( r) of 0.9996. The lower limit of quantification (LLOQ) was identifiable and reproducible at 0.5 ng/ml. The validated method was successfully applied to study the pharmacokinetics of cymipristone in healthy Chinese female subjects.

Sensitive and rapid method for amino acid quantitation in malaria biological samples using AccQ.Tag ultra performance liquid chromatography-electrospray ionization-MS/MS with multiple reaction monitoring.

An AccQ*Tag ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (AccQ*Tag-UPLC-ESI-MS/MS) method for fast, reproducible, and sensitive amino acid quantitation in biological samples, particularly, the malaria parasite Plasmodium falciparum is presented. The Waters Acquity TQD UPLC/MS system equipped with a photodiode array (PDA) detector was used for amino acid separation and detection. The method was developed and validated using amino acid standard mixtures containing acidic, neutral, and basic amino acids. For MS analysis, the optimum cone voltage implemented, based on direct infusion analysis of a few selected AccQ*Tag amino acids with multiple reaction monitoring, varied from 29 to 39 V, whereas the collision energy varied from 15 to 35 V. Calibration curves were built using both internal and external standardization. Typically, a linear response for all amino acids was observed at concentration ranges of 3 x 10(-3)-25 pmol/muL. For some amino acids, concentration limits of detection were as low as 1.65 fmol. The coefficients of variation for retention times were within the range of 0.08-1.08%. The coefficients of variation for amino acid quantitation, determined from triplicate UPLC-MS/MS runs, were below 8% on the average. The developed AccQ*Tag-UPLC-ESI-MS/MS method revealed good technical and biological reproducibility when applied to P. falciparum and human red blood cells samples. This study should provide a valuable insight into the performance of UPLC-ESI-MS/MS for amino acid quantitation using AccQ*Tag derivatization.

Quantitative LC-ESI-MS/MS metabolic profiling method for fatty acids and lipophilic metabolites in fermentation broths from β-lactam antibiotics production

In the present paper, we report on the development of a straightforward reversed-phase liquid chromatography-electrospray ionization-tandem mass spectrometry method for the determination of the most abundant fatty acids; alpha-tocopherol and cephalosporin P1 in fermentation broths. Using this method, fatty acids could be successfully determined in extracts of fermentation broths from penicillin and cephalosporin production without prior derivatization. Matrix effects were investigated in detail, and various kinds of calibrations (i.e., by use of neat standard solutions as well as by matrix-matched calibration employing standard addition each with and without internal standards) were comparatively assessed. The optimized and validated method was employed for the analysis of extracts of fermentation broths and nutrition media.

Development and Validation of a High-Performance Liquid Chromatography—Tandem Mass Spectrometry Method for the Rapid Simultaneous Quantification of Aconitine, Mesaconitine, and Hypaconitine in Rat Plasma after Oral Administration of Sini Decoction

A rapid, sensitive, and specific liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) method was developed and validated for simultaneous determination of aconitine (AC), mesaconitine (MA), and hypaconitine (HA), the three toxic constituents from Sini decoction (SND) in rat plasma. After the addition of citalopram as the internal standard (IS), plasma samples were basified with 100 microL 10% ammonium hydroxide, and then extracted with 1 mL ethyl acetate. Chromatographic separation was performed on a CN column (250 mm x 4.6 mm, 5 microm) with a mobile phase of methanol/40 mM ammonium acetate/formic acid (950:45:5, v/v/v) at the flow rate of 1.0 mL/min. Analytes were determined in a triple-quadrupole mass spectrometer in the selected reaction-monitoring (SRM) mode using electrospray source with positive mode. The method was validated over the concentration ranges of 0.01-10 ng/mL for AC, MA, and HA. The variation coefficients were always < 15% for both intraday and interday precision for each analyte. Mean accuracies were also within +/-15%. The method was proved to be sensitive, rapid, specific, accurate, and reproducible. It has been successfully applied to the pharmacokinetics study on rats after oral administration of SND.

Oxycodone-Related Fatalities in the West of Scotland

The intent of this study was to review fatalities involving oxycodone in the west of Scotland using a liquid chromatography-electrospray ionization-tandem mass spectrometry method developed for the determination of oxycodone and N- and O-demethylated metabolites in unhydrolyzed postmortem specimens. Ten oxycodone positive postmortem cases were detected, and nine were drug-related fatalities. Five cases were attributed solely to oxycodone intoxication and four to polydrug intoxication. Although there was overlap between blood oxycodone levels in deaths attributed to oxycodone only and those due to polydrug intoxication, lower oxycodone levels (< 1 mg/L) were associated with polydrug intoxication when compared with cases due to oxycodone alone (> 1 mg/L). The role of the parent drug in oxycodone fatalities has been fully studied, but the role of oxycodone metabolites (noroxycodone and oxymorphone) was investigated in this report for the first time. Oxycodone was more commonly detected in blood, urine, and vitreous humor followed by noroxycodone. The ratio between oxycodone and its N-demethylated metabolite was evaluated and found to be useful in determining whether death occurred shortly after drug administration or if there was a significant delay. High parent/metabolite ratios were correlated with short survival times after ingestion. The median ratio of oxycodone/noroxycodone was 2.4 and ranged from 0.7 to 49. Oxycodone prescriptions have risen sharply in Scotland in recent years, and the identification of 10 oxycodone-related deaths in the past 18 months highlights the importance of including this drug in routine laboratory screening and confirmation procedures.

Simultaneous quantification of oleuropein and its metabolites in rat plasma by liquid chromatography electrospray ionization tandem mass spectrometry

Oleuropein (OE) is the cardinal bioactive compound derived from Olea europaea and possesses numerous beneficial properties for human health. However, despite the plethora of analytical methods that have studied the biological fate of olive oil-derived bioactive compounds, no validated methodology has been published to date for the simultaneous determination of OE, along with all its major metabolites. In this study, a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI MS/MS) method has been developed and validated for the quantification of OE, simultaneously with its main metabolites hydroxytyrosol, 2-(3,4-dihydroxyphenyl)acetic acid, 4-(2-hydroxyethyl)-2-methoxy-phenol or homovanillyl alcohol, 2-(4-hydroxy-3-methoxyphenyl)acetic acid or homovanillic acid, and elenolic acid in rat plasma matrix. Samples were analyzed by LC-ESI MS/MS prior to and after enzymatic treatment. A solid-phase extraction step with high mean recovery for all compounds was performed as sample pretreatment. Calibration curves were linear for all bioactive compounds over the range studied, while the method exhibited good accuracy, intra- and inter-day precision. The limit of detection was in the picogram range (per milliliterof plasma) for HT and OE and in the nanogram range (per milliliter of plasma) for the other analytes, and the method was simple and rapid. The developed methodology was successfully applied for the simultaneous quantification of OE and its aforementioned metabolites in rat plasma samples, thus demonstrating its suitability for pharmacokinetics, as well as bioavailability and metabolism studies.

The Determination of Haloacetic Acids in Real World Samples using IC-ESI-MS-MS

This paper presents the determination of nine haloacetic acids (HAAs) in high ionic strength, treated effluent waters using an ion chromatography-electrospray ionization-tandem mass spectrometry (IC-ESI-MS-MS) method with internal standards and discussions of each of the method parameters. Data is also provided for these same samples using USEPA Method 552.2. The sample matrices contain up to 170 mg/L chloride and 243 mg/L sulfate. Matrix ions are separated from the analytes using a high capacity anion exchange analytical column and diverted to a waste stream during each analysis to avoid signal suppression and contamination of the detector. No derivatization, offline matrix elimination, or preconcentration is used. Four isotopically-labeled HAAs are used for quantification, and detection limits are in the range of 400-1000 microg/L with R(2) of at least 0.997 over two orders of magnitude for all analytes in matrix. A trichloroacetic acid (TCAA) internal standard with the label on the alpha carbon is found to be more stable than the TCAA-1-(13)C. Amounts found using IC-MS-MS are 65-130% of amounts found using Method 552.2 for all analytes in the real world treated effluent waters. Detection limits for all nine analytes in matrix are in the range of 100-700 ng/L.

Identification of the Metabolites of Ecabet Bismuth in Rat Bile by Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry

In the present study, a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI- MS/MS) method was developed for the screening and the structural elucidation of the metabolites of ecabet bismuth in rat bile. Solid-phase extraction cartridges were used for sample pre-treatment and a gradient liquid chromatographic system composed of 10 mM ammonium acetate buffer and methanol was used for chromatographic separation on a Phenomenex Kromasil C(18) column. The triple quadrupole mass spectrometer was employed to thoroughly detect and acquire the detailed MS/MS spectra of ecabet and its metabolites. By comparing the chromatographic retention behaviors, as well as the changes in molecular weight and full-scan MS/MS spectra of the potential metabolites with those of the parent compound, two main metabolites were identified as glucuronide conjugate of carbonylated ecabet (7-oxo-ecabet) and glucuronide conjugate of ecabet. Both two metabolites have not been reported in the literatures. The metabolic pathways of ecabet in rat were also proposed in this paper.

A liquid chromatography/tandem mass spectrometry method for determination of aristolochic acid‐I in rat plasma

A sensitive, rapid and specific liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed and validated for the determination of aristolochic acid-I (AA-I) in rat plasma. Finasteride was used as the internal standard (IS). The analyte was separated on a Zorbax Eclipse XDB-C(18) column by isocratic elution with methanol-10 mM ammonium acetate (75:25, v/v, pH = 7.3) at a flow rate of 0.25 mL/min, and analyzed by mass spectrometry in positive multiple reaction monitoring mode. The precursor-to-product ion transitions of m/z 359.0 --> 298.2 and m/z 373.1 --> 305.2 were used to detect AA-I and IS, respectively. Good linearity was achieved over a range of 0.4-600 ng/mL. Intra- and inter-day precisions measured as relative standard deviation were less than 13.5%, and accuracy ranged from 94.2 to 97.5%. The developed method was successfully applied in the pharmacokinetic study of AA-I in rats.

Determination of dexmedetomidine in human plasma using high performance liquid chromatography coupled with tandem mass spectrometric detection: Application to a pharmacokinetic study

A rapid, sensitive and selective high performance liquid chromatography-electrospray ionization-tandem mass spectrometry method (HPLC-ESI-MS/MS) was developed and validated for the determination and pharmacokinetic investigation of dexmedetomidine (DMED) in human plasma. Dexmedetomidine and the internal standard (ondansetron) were extracted in a single step with diethyl-ether from 1.0 mL of alkalinized plasma. The mobile phase was a mixture of acetonitrile and 0.5% formic acid solution (30:70, v/v) at a flow rate of 0.2 mL min −1. The detection was performed on a triple quadrupole tandem mass spectrometer in the selected reaction monitoring (SRM) mode using the respective [M+H] + ions m/z 201.0 → 95.1 for DMED and m/z 294.1 → 170.1 for the IS. The assay exhibited a linear dynamic range of 5–5000 pg mL −1 with the correlation coefficient above 0.9995. The lower limit of quantification (LLOQ) was 5 pg mL −1 with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The validated HPLC-MS/MS method has been successfully applied to study the pharmacokinetics of three level doses of DMED in Chinese healthy volunteers.

Application of a liquid chromatography/tandem mass spectrometry method for the pharmacokinetic study of dihydroartemisinin injectable powder in healthy Chinese subjects

A simple, rapid and accurate liquid chromatography–electrospray ionization-tandem mass spectrometry method was developed and validated for quantification of dihydroartemisinin (DHA) in human plasma. Following a simple single-step liquid–liquid extraction with ethyl acetate, the analyte was separated on a C 18 column by isocratic elution with methanol–water–10 mM ammonium acetate (80:10:10, v/v/v), and analyzed by mass spectrometry in the positive ion MRM mode. Good linearity was achieved over a wide range of 1.01–2020 ng/mL. Intra- and inter-day precisions were less than 9.0%, and accuracy ranged from 93.0 to 98.2%. The pharmacokinetics of DHA injectable powder was studied for the first time in healthy subjects by this method. After single intravenous infusion of DHA injectable powder 40, 80 and 160 mg, the elimination half-life ( t 1/2 λZ ) was 1.69, 1.88 and 1.92 h, respectively; mean C max and AUC increased in proportion to the doses. The pharmacokinetics of DHA fit the linear dynamic feature over the DHA dose range studied.

A Sensitive LC–ESI–MS–MS Method for the Determination of Huperzine A in Human Plasma: Method and Clinical Applications

A sensitive and specific liquid chromatography–electrospray ionization–tandem mass spectrometry method has been developed and validated for the quantification of huperzine A in human plasma. After the addition of trimetazidine, the internal standard (IS) and sodium hydroxide, plasma samples were extracted using 5 mL ethyl acetate. The compounds were separated on an Agilent Zorbax SB C18 column (100 mm × 2.1 mm ID, dp 3.5 μm) using an elution system of 10 mM ammonium acetate solution–methanol–formic acid (18:82:0.1, v/v) as the mobile phase. The quantification of target compounds was obtained by using multiple reaction monitoring (MRM) transitions: m/z 243.1, 210.1 and 267.2, 166.0 were measured in positive mode for huperzine A and IS. Linearity was established for the range of concentrations 0.01–4.0 ng mL−1 with a coefficient of correlation (r) of 0.9991. The lower limit of quantification (LLOQ) was identifiable and reproducible at 0.01 ng mL−1. The method has been successfully applied to study the pharmacokinetics of huperzine A in healthy male Chinese volunteers.

A fully validated isotope dilution HPLC‐MS/MS method for the simultaneous determination of succinylcholine and succinylmonocholine in serum and urine samples

A high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method for the simultaneous detection of succinylcholine (SUX) and its metabolite succinylmonocholine (SMC) in serum and urine is presented. For internal standardization using isotope dilution, the deuterated compounds SUX-d(18) and SMC-d(3) were employed. Full validation was performed according to international guidelines. Solid-phase extraction (SPE) of acidified samples was accomplished using Strata-X polymeric reversed phase cartridges together with heptafluorobutyric acid (HFBA) as ion-pairing reagent. Separation was achieved within 13 min on a Phenomenex Synergi Hydro RP C18 column (4 microm, 150 x 2 mm) using a gradient of 5 mM ammonium formate buffer pH 3.5 and acetonitrile.To ensure the method's applicability in forensic as well as clinical toxicology, the specific demands of both research fields were taken into account, and the method was thus validated for a low and high concentration range. For both serum and urine as sample matrix, the validation revealed good intraday and interday precisions, consistently ranging below 15% for the lowest and below 10% for elevated concentrations. Accuracy was likewise good and never exceeded 10%. Extraction recovery was excellent, ranging between 88.1 and 103.9% for SUX and SMC in both tested matrices. Matrix effects were significant, the otherwise optimized extraction and detection methods, however, allowed for a very satisfactory sensitivity of the described method: For serum, the limits of detection and quantitation were determined to be 1.9 and 6.0 ng/ml for SUX, as well as 2.5 and 8.6 ng/ml for SMC, respectively; for urine, the corresponding values were established to be 1.4 and 4.0 ng/ml (SUX), as well as 1.5 and 4.9 ng/ml (SMC).The presented method was successfully applied to authentic samples of two forensic cases investigated in the institute of forensic medicine in Bonn, allowing the diagnosis of SUX intoxications.

An Enantiomer-Selective Liquid Chromatography-Tandem Mass Spectrometry Method for Methadone and EDDP Validated for Use in Human Plasma, Urine, and Liver Microsomes

A liquid chromatography-electrospray ionization-tandem mass spectrometry method has been developed and validated to detect (R)- and (S)-methadone and (R)- and (S)-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in human plasma with cross-validation to urine and liver microsomes. Use of deuterated internal standards and liquid-liquid extraction coupled with chiral separation provided baseline separation with a lower limit of quantitation (LLOQ) of 2.5 ng/mL. The LLOQ was established from comparison of signal in blanks from six different sources per matrix with the same sources fortified at the LLOQ (none exceeded 19% of LLOQ) and precision and accuracy at the LLOQ determined in the same six sources per matrix. The assay was precise (% coefficients of variation within 13.8%) and accurate (% targets within 15%) in all three matrices. No interference was seen from addition of other psychoactive drugs. Stability was determined in plasma (24 h at room temperature, 321 days at -20 degrees C, 3 freeze-thaw cycles); processed plasma samples (5 days at -20 degrees C, 12 days on autosampler); urine (24 h at room temperature); and stock solutions (20 h at room temperature, 61 days at -20 degrees C). Applications of varying degree are presented for each matrix. Plasma from five subjects maintained on 100 mg oral methadone per day permitted comparison of the pharmacokinetics of the enantiomers. The t(1/2) of (R)-methadone was significantly longer than for (S)-methadone, and (S)-methadone was more tightly protein bound. The C(max), AUC, C(min), and % protein bound of (S)-EDDP were significantly greater than (R)-EDDP, while the t(1/2) of (R)-EDDP was significantly greater than (S)-EDDP. In spot urines, (R)- was higher than (S)-methadone, and (S)- was generally higher than (R)-EDDP. (R)- and (S)-EDDP production was detected after incubation of therapeutic concentrations of racemic methadone with human liver microsomes, and (S)-EDDP production was twofold greater than (R)-EDDP in three human placental microsomes incubated with racemic methadone.