Development and validation of a multi‐analyte LC‐MS/MS approach for quantification of neuroleptics in whole blood,plasma, and serum

Based on a similar approach for quantification of antidepressants, benzodiazepines, and z‐drugs, a liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) multi‐analyte approach with simple liquid‐liquid extraction was extended for fast target screening and quantification of neuroleptics in whole blood, plasma, and serum. As this method is part of a multi‐analyte procedure for over 100 analytes from different drug classes and as the extracts were additionally used in the authors' laboratory for gas chromatography‐mass spectrometry (GC‐MS) analysis, one universal stable‐isotope‐labelled internal standard (SIL‐IS) was used to save time and resource. The method was validated with respect to international guidelines. For accuracy and precision, full calibration was performed with ranges from subtherapeutic to toxic concentrations. Selectivity problems could not be observed, but matrix effects ranged from 68 to 211% in all samples. For the low quality control (QC), recovery ranged from 32 to 112%, process efficiency from 31 to 165% and for the high QC recovery from 42 to 141%, process efficiency from 29 to 154%. In addition statistical data evaluation of the variances of the recovery, matrix effects, and process efficiency data between whole blood vs. plasma, whole blood vs. serum, and plasma vs. serum were done. The presented LC‐MS/MS approach was applicable for selective detection of 33 neuroleptics as well as accurate and precise quantification of 25 neuroleptics in whole blood, 19 in plasma, and 17 in serum. More significant matrix effects (ME) for neuropletic drugs overall in plasma and serum as compared with whole blood were detected. Copyright © 2015 John Wiley & Sons, Ltd.     

Development and validation of an LC‐MS/MS method after chiral derivatization for the simultaneous stereoselective determination of methylenedioxy‐methamphetamine (MDMA) and its phase I and II metabolites in human blood plasma

3,4‐Methylenedioxymethamphetamine (MDMA, ecstasy) is a racemic drug of abuse and its two enantiomers are known to differ in their dose‐response curves. The S‐enantiomer was shown to be eliminated at a higher rate than the R‐enantiomer. The most likely explanation for this is a stereoselective metabolism also claimed in in vitro studies. Urinary excretion studies showed that the main metabolites in humans are 4‐hydroxy 3‐methoxymethamphetamine (HMMA) 4‐sulfate, HMMA 4‐glucuronide and 3,4‐dihydroxymethamphetamine (DHMA) 3‐sulfate. For stereoselective pharmacokinetic analysis of phase I and phase II metabolites in human blood plasma useful analytical methods are needed. Therefore the aim of the presented study was the development and validation of a stereoselective liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method for the simultaneous quantification of MDMA, 3,4‐methylenedioxyamphetamine, DHMA, DHMA 3‐sulfate, HMMA, HMMA 4‐glucuronide, HMMA 4‐sulfate, and 4‐hydroxy 3‐methoxyamphetamine in blood plasma for evaluation of the stereoselective pharmacokinetics in humans. Blood plasma samples were prepared by simple protein precipitation and afterwards all analytes were derivatized using N‐(2,4‐dinitro‐5‐fluorophenyl) L‐valinamide resulting in the formation of diastereomers which were easily separable on standard reverse phase stationary phases. This simple and fast method was validated according to international guidelines including specificity, recovery, matrix effects, accuracy and precision, stabilities, and limits of quantification. The method proved to be selective, sensitive, accurate and precise for all tested analytes except for DHMA. Copyright © 2014 John Wiley & Sons, Ltd.     

Elucidation of the metabolites of the novel psychoactive substance 4‐methyl‐N‐ethyl‐cathinone (4‐MEC) in human urine and pooled liver microsomes by GC‐MS and LC‐HR‐MS/MS techniques and of its detectability by GC‐MS or LC‐MSn standard screening approaches

4‐methyl‐N‐ethcathinone (4‐MEC), the N‐ethyl homologue of mephedrone, is a novel psychoactive substance of the beta‐keto amphetamine (cathinone) group. The aim of the present work was to study the phase I and phase II metabolism of 4‐MEC in human urine as well as in pooled human liver microsome (pHLM) incubations. The urine samples were worked up with and without enzymatic cleavage, the pHLM incubations by simple deproteinization. The metabolites were separated and identified by gas chromatography‐mass spectrometry (GC‐MS) and liquid chromatography‐high resolution‐tandem mass spectrometry (LC‐HR‐MS/MS). Based on the metabolites identified in urine and/or pHLM, the following metabolic pathways could be proposed: reduction of the keto group, N‐deethylation, hydroxylation of the 4‐methyl group followed by further oxidation to the corresponding 4‐carboxy metabolite, and combinations of these steps. Glucuronidation could only be observed for the hydroxy metabolite. These pathways were similar to those described for the N‐methyl homologue mephedrone and other related drugs. In pHLM, all phase I metabolites with the exception of the N‐deethyl‐dihydro isomers and the 4‐carboxy‐dihydro metabolite could be confirmed. Glucuronides could not be formed under the applied conditions. Although the taken dose was not clear, an intake of 4‐MEC should be detectable in urine by the GC‐MS and LC‐MSⁿ standard urine screening approaches at least after overdose. Copyright © 2014 John Wiley & Sons, Ltd.     

Lefetamine‐derived designer drugs N‐ethyl‐1,2‐diphenylethylamine (NEDPA) and N‐iso‐propyl‐1,2‐diphenylethylamine (NPDPA): Metabolism and detectability in rat urine using GC‐MS,LC‐MSn and LC‐HR‐MS/MS

N‐Ethyl‐1,2‐diphenylethylamine (NEDPA) and N‐iso‐propyl‐1,2‐diphenylethylamine (NPDPA) are two designer drugs, which were confiscated in Germany in 2008. Lefetamine (N,N‐dimethyl‐1,2‐diphenylethylamine, also named L‐SPA), the pharmaceutical lead of these designer drugs, is a controlled substance in many countries. The aim of the present work was to study the phase I and phase II metabolism of these drugs in rats and to check for their detectability in urine using the authors’ standard urine screening approaches (SUSA). For the elucidation of the metabolism, rat urine samples were worked up with and without enzymatic cleavage, separated and analyzed by gas chromatography‐mass spectrometry (GC‐MS) and liquid chromatography‐high resolution‐tandem mass spectrometry (LC‐HR‐MS/MS). According to the identified metabolites, the following metabolic pathways for NEDPA and NPDPA could be proposed: N‐dealkylation, mono‐ and bis‐hydroxylation of the benzyl ring followed by methylation of one of the two hydroxy groups, combinations of these steps, hydroxylation of the phenyl ring after N‐dealkylation, glucuronidation and sulfation of all hydroxylated metabolites. Application of a 0.3 mg/kg BW dose of NEDPA or NPDPA, corresponding to a common lefetamine single dose, could be monitored in rat urine using the authors’ GC‐MS and LC‐MSⁿ SUSA. However, only the metabolites could be detected, namely N‐deethyl‐NEDPA, N‐deethyl‐hydroxy‐NEDPA, hydroxy‐NEDPA, and hydroxy‐methoxy‐NEDPA or N‐de‐iso‐propyl‐NPDPA, N‐de‐iso‐propyl‐hydroxy‐NPDPA, and hydroxy‐NPDPA. Assuming similar kinetics, an intake of these drugs should also be detectable in human urine. Copyright © 2014 John Wiley & Sons, Ltd.     

Toxicokinetics of new psychoactive substances: plasma protein binding,metabolic stability,and human phase I metabolism of the synthetic cannabinoid WIN 55,212‐2 studied using in vitro tools and LC‐HR‐MS/MS

The new psychoactive substance WIN 55,212‐2 ((R)‐(+)‐[2,3‐dihydro‐5‐methyl‐3‐(4‐morpholinylmethyl)pyrrolo‐[1,2,3‐de]‐1,4‐benzoxazin‐6‐yl]‐1‐napthalenylmethanone) is a potent synthetic cannabinoid receptor agonist. The metabolism of WIN 55,212‐2 in man has never been reported. Therefore, the aim of this study was to identify the human in vitro metabolites of WIN 55,212‐2 using pooled human liver microsomes and liquid chromatography‐high resolution‐tandem mass spectrometry (LC‐HR‐MS/MS) to provide targets for toxicological, doping, and environmental screening procedures. Moreover, a metabolic stability study in pooled human liver microsomes (pHLM) was carried out. In total, 19 metabolites were identified and the following partly overlapping metabolic steps were deduced: degradation of the morpholine ring via hydroxylation, N‐ and O‐dealkylation, and oxidative deamination, hydroxylations on either the naphthalene or morpholine ring or the alkyl spacer with subsequent oxidation, epoxide formation with subsequent hydrolysis, or combinations. In conclusion, WIN 55,212‐2 was extensively metabolized in human liver microsomes incubations and the calculated hepatic clearance was comparably high, indicating a fast and nearly complete metabolism in vivo. This is in line with previous findings on other synthetic cannabinoids. Copyright © 2016 John Wiley & Sons, Ltd.     

液相色谱-质谱联用法测定人血浆双氢青蒿素浓度

目的:建立液相色谱-质谱联用法测定健康人血浆中双氢青蒿素浓度的方法。方法:以青蒿素为内标,血浆样品采用液-液萃取法处理。用电喷雾离子化和正离子多离子反应监测方式检测双氢青蒿素。结果:该方法双氢青蒿素线性范围为1.01~2020 ng.ml-1;定量下限为1.001±0.072 ng.ml-1;方法回收率在93.0%~98.2%;批内、批间变异系数均<10%。结论:该方法准确、灵敏、特异、简便,适用于健康人血浆双氢青蒿素浓度的测定。     

LC‐MS/MS detection of unaltered glucuronoconjugated metabolites of metandienone

The aim of this study was to evaluate the direct detection of glucuronoconjugated metabolites of metandienone (MTD) and their detection times. Metabolites resistant to enzymatic hydrolysis were also evaluated. Based on the common mass spectrometric behaviour of steroid glucuronides, three liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) strategies were applied for the detection of unpredicted and predicted metabolites: precursor ion scan (PI), neutral loss scan (NL), and theoretical selected reaction monitoring (SRM) methods. Samples from four excretion studies of MTD were analyzed for both the detection of metabolites and the establishment of their detection times. Using PI and NL methods, seven metabolites were observed in post‐administration samples. SRM methods allowed for the detection of 13 glucuronide metabolites. The detection times, measured by analysis with an SRM method, were between 1 and 22 days. The metabolite detected for the longest time was 18‐nor‐17β‐hydroxymethyl‐17α‐methyl‐5β‐androsta‐1,4,13‐triene‐3‐one‐17‐glucuronide. One metabolite was resistant to hydrolysis with β ‐glucuronidase; however it was only detected in urine up to four days after administration. The three glucuronide metabolites with the highest retrospectivity were identified by chemical synthesis or mass spectrometric data, and although they were previously reported, this is the first time that analytical data of the intact phase II metabolites are presented for some of them. The LC‐MS/MS strategies applied have demonstrated to be useful for detecting glucuronoconjugated metabolites of MTD, including glucuronides resistant to enzymatic hydrolysis which cannot be detected by conventional approaches. Copyright © 2016 John Wiley & Sons, Ltd.     

Application of an LC‐MS/MS method to the pharmacokinetics of TM‐2, a potential antitumour agent,in rats

TM‐2 is a novel semi‐synthetic taxane derivative, selected for preclinical development based on its greater anticancer activity and lower toxicity compared with docetaxel. In this study, a rapid and sensitive analytical method based on ultra performance liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) has been developed for the determination of TM‐2 in rat plasma. The biological samples were extracted with methyl tert‐butyl ether and separated on a C₁₈ column (50 mm × 2.1 mm, 1.7 µm) using a mobile phase consisting of acetonitrile and 2 mM ammonium acetate. The standard curves were linear over the range 5–1000 ng/mL in rat plasma. The precision (relative standard deviation, RSD, %) were within 14.5%, and the accuracy (relative error, RE, %) ranged from ?1.56 to 2.36%. Recovery and matrix effect were satisfactory in rat plasma. The validated method was successfully applied to pharmacokinetic studies after intravenous administration of TM‐2 to rats. The pharmacokinetics of TM‐2 in rats were characterized by a large volume of distribution and a long half‐life of elimination after single dose (4, 8, and 16 mg/kg), and a good correlation was observed between AUC and dose. The preclinical data will be useful for the design of subsequent trials of TM‐2. Copyright © 2014 John Wiley & Sons, Ltd.     

Development and validation of an ESI‐LC‐MS/MS method for simultaneous identification and quantification of 24 analytes of forensic relevance in vitreous humour,whole blood and plasma

Detection and quantification of drugs from various biological matrices are of immense importance in forensic toxicological analysis. Despite the various reported methods, development of a new method for the detection and quantification of drugs is still an active area of research. However, every method and biological matrix has its own limitation, which further encourage forensic toxicologists to develop new methods and to explore new matrices for the analysis of drugs. In this study, an electrospray ionization‐liquid chromatograph‐tandem mass spectrometry (ESI‐LC‐MS/MS) method is developed and validated for simultaneous identification and quantification of 24 drugs of forensic relevance in various body fluids, namely, whole blood, plasma and vitreous humour. The newly developed method has been validated for intra‐day and inter‐day accuracy, precision, selectivity and sensitivity. Absolute recovery shows a mean of 84.5, 86.2, and 103% in the vitreous humour, whole blood and plasma respectively, which is suitable for the screening procedure. Further, the absolute matrix effect (AME) shows a mean of 105, 96.5, and 109% in the vitreous humour, whole blood and plasma, respectively. In addition, to examine the practical utility of this method, it has been applied for screening of drugs in post‐mortem samples of the vitreous humour, whole blood and plasma collected at autopsy from ten cadavers. Experimental results show that the newly developed method is well applicable for screening of analytes in all the three matrices. Copyright © 2015 John Wiley & Sons, Ltd.     

液相色谱-质谱联用法测定人参皂苷Re在健康人血浆的浓度

目的建立液相色谱-质谱联用法测定人参皂苷Re在健康人血浆中浓 度的方法。方法血浆样品用固相萃取法处理。用电喷雾离子化和正离子多 离子反应监测方式检测人参皂苷Re。结果该方法人参皂苷Re线性范围为 1．05～1 050 ng·mL-1;定量下限为1．05 ng·mL-1;方法回收率在99．3％～ 104．3％;日内、日间变异系数(RSD)均<15％。结论该法准确、灵敏、特异, 适用于健康人血浆人参皂苷Re浓度测定。     

Development and validation of an LC‐MS/MS method for simultaneous determination of piperaquine and 97‐63, the active metabolite of CDRI 97‐78, in rat plasma and its application in interaction study

Piperaquine‐dihydroartemisinin combination is the latest addition to the repertoire of ACTs recommended by the World Health Organization (WHO) for treatment of falciparum malaria. Due to the increasing resistance to artemisinin derivatives, CSIR‐CDRI has developed a prospective short acting, trioxane antimalarial derivative, CDRI 97‐78. In the present study, a liquid chromatography‐electrospray ionization‐tandem mass spectrometry (LC–ESI‐MS/MS) method for the simultaneous quantification of piperaquine (PPQ) and 97‐63, the active metabolite of CDRI 97‐78 found in vivo, was developed and validated in 100 μL rat plasma using halofantrine as internal standard. PPQ and 97‐63 were separated using acetonitrile:methanol (50:50, v/v) and ammonium formate buffer (10 mM, pH 4.5) in the ratio of 95:5(v/v) as mobile phase under isocratic conditions at a flow rate of 0.65 mL/min on Waters Atlantis C₁₈ (4.6 × 50 mm, 5.0 µm) column. The extraction recoveries of PPQ and 97‐63 ranged from 90.58 to 105.48%, while for the internal standard, it was 94.27%. The method was accurate and precise in the linearity range 3.9–250 ng/mL for both the analytes, with a correlation coefficient (r) of ≥ 0.998. The intra‐ and inter‐day assay precision ranged from 2.91 to 8.45% and; intra‐ and inter‐day assay accuracy was between 92.50 and 110.20% for both the analytes. The method was successfully applied to study the effect of oral co‐administration of PPQ on the pharmacokinetics of CDRI 97‐78 in Sprague‐dawley rats and vice versa. The co‐administration of CDRI 97‐78 caused significant decrease in AUC_0–∞ of PPQ from 31.52 ± 2.68 to 14.84 ± 4.33 h*µg/mL. However, co‐administration of PPQ did not have any significant effect on the pharmacokinetics of CDRI 97‐78. Copyright © 2015 John Wiley & Sons, Ltd.     

A method for confirming CJC‐1295 abuse in equine plasma samples by LC–MS/MS

CJC‐1295 is a peptide‐based drug that stimulates the production of growth hormone (GH) from the pituitary gland. It incorporates a functional maleimido group at the C‐terminus that allows it to covalently bind plasma proteins such as serum albumin. These CJC‐1295‐protein conjugates have a much greater half‐life compared to the unconjugated peptide and are capable of stimulating GH production for more than six days in humans after a single administration. Conjugated CJC‐1295 is difficult to detect in blood by mass spectrometry due to its low abundance, high molecular weight, and conjugation to a range of different protein substrates. Previously we described a screening procedure for the detection of CJC‐1295 in equine plasma using an immuno‐PCR assay. Here we demonstrate the confirmation of CJC‐1295 in equine plasma by LC?MS/MS after immuno‐affinity capture and tryptic digestion. Using this method, CJC‐1295 was identified down to concentrations as low as 180 pg/mL in 1 mL of equine plasma.     

The in vivo and in vitro phase I metabolism of FYL‐67, a novel oxazolidinone antibacterial drug,studied by LC‐MS/MS

In our previous study, FYL‐67, a novel linezolid analogue with the morpholinyl ring replaced by a 4‐(pyridin‐2‐yl)‐1H‐pyrazol‐1‐yl group, was demonstrated to own an excellent activity against Gram‐positive organisms,such as methicillin‐resistant Staphylococcus aureus (MRSA). However, metabolic biotransformation was not investigated. This study was performed to identify the phase I metabolites of FYL‐67 using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). The chemical structures were confirmed by comparison with corresponding chemical standards obtained internal. Primary elucidation of the metabolic pathway of FYL‐67 in vitro was performed using liver preparations (microsomes and hepatocytes) from rats and humans, and SD (Sprague Dawley, rat, rattus norvegicus) rats were used for the study of in vivo approach. To the end, two metabolites (M₁ and M₂) were detected after in vitro as well as in vivo experiments. Based on LC‐MS/MS analyses, the metabolites were demonstrated to be 5‐(aminomethyl)‐3‐(3‐fluoro‐4‐(4‐(pyridin‐2‐yl)‐1H‐pyrazol‐1‐yl)phenyl)oxazolidin‐2‐one (M₁) and 3‐(3‐fluoro‐4‐(4‐(pyridin‐2‐yl)‐1H‐pyrazol‐1‐yl)phenyl)‐5‐(hydroxymethyl)oxazolidin‐2‐one (M₂). Amide hydrolysis at acetyl group of FYL‐67 leading to the formation of M₁ was observed and suggested to play a major role in both in vivo and in vitro phase I metabolism of FYL‐67. M₁ was demonstrated to undergo a further oxidation to form M₂. In addition, the results indicated no species difference existing between rats and humans. The outcomes of our research can be utilized for the development and validation of the analytical method for the quantification of FYL‐67 as well as its metabolites in biological samples. Furthermore, it is helpful to conduct studies of pharmacodynamics and toxicodynamics. Copyright © 2015 John Wiley & Sons, Ltd.     

A simple extraction and LC‐MS/MS approach for the screening and identification of over 100 analytes in eight different matrices

In the present study, a liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) multi‐analyte approach using one single work‐up approach in whole blood, plasma, serum, post‐mortem blood, liver tissue, gastric content, hair, and urine was developed for fast target screening and reliable identification of 130 analytes often requested in clinical and forensic toxicology. Samples (500 μL each) of whole blood, plasma, serum, post‐mortem blood, tissue (homogenized 1 + 4 with water), as well as 3 g of distilled gastric contents, 1 mL of urine, or 20 mg of pulverized hair were extracted at different pH values with an diethyl ether‐ethyl acetate mixture (1:1). Separation and identification were performed using LC‐QTRAP with electrospray ionization in positive mode. For identification 1 scheduled multi‐reaction‐mode (sMRM) method with 390 transitions was developed covering benzodiazepines, Z‐drugs, antidepressants, neuroleptics, opioids, new synthetic drugs, and phosphodiesterase type 5 inhibitors. For positive sMRM transitions with intensities exceeding 5000 cps, dependent scans (EPI scan collision energy, 35 eV, collision energy spread, 15 eV) were performed for library search using our in‐house library. The method was developed with respect to selectivity, matrix effects, recovery, process efficiency, limit of detection, and applicability. The simple work‐up procedure was suitable for all biosamples with exception of urine in respect to low concentrated analytes, which showed median recovery values of 59%. The method was selective for 130 analytes in all 8 biosamples. For 106 analytes, the limit of detection in whole blood, plasma, and serum was lower than the lowest therapeutic concentration listed in blood level lists. Copyright © 2014 John Wiley & Sons, Ltd.     

MALDI MSI and LC‐MS/MS: Towards preclinical determination of the neurotoxic potential of fluoroquinolones

Fluoroquinolones are broad‐spectrum antibiotics with efficacy against a wide range of pathogenic microbes associated with respiratory and meningeal infections. The potential toxicity of this class of chemical agents is a source of major concern and is becoming a global issue. The aim of this study was to develop a method for the brain distribution and the pharmacokinetic profile of gatifloxacin in healthy Sprague‐Dawley rats, via Multicenter matrix‐assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) and quantitative liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). We developed a sensitive LC‐MS/MS method to quantify gatifloxacin in plasma, lung, and brain homogenates. A pharmacokinetic profile was observed where there is a double peak pattern; a sharp initial increase in the concentration soon after dosing followed by a steady decline until another increase in concentration after a longer period post dosing in all three biological samples was observed. The imaging results showed the drug gradually entering the brain via the blood brain barrier and into the cortical regions from 15 to 240 min post dose. As time elapses, the drug leaves the brain following the same path as it followed on its entry and finally concentrates at the cortex. Copyright © 2015 John Wiley & Sons, Ltd.     

Synthesis,radio‐LC‐MS analysis and biological evaluation of 99mTc‐techmazenil

A ^99mTc‐labelled compound with the biological characteristics of flumazenil would be useful for determination of neuronal viability after the onset of a stroke. Therefore, we have derivatized Ro‐15‐3890 (a flumazenil metabolite bearing a carboxylic acid group instead of an ethyl ester) by coupling it with a bisamino bisthiol tetraligand bearing a 3‐hydroxypropyl side chain (3‐hydroxypropyl‐BAT) to enable labelling with technetium‐99m. After purification by RP‐HPLC, the ligand was deprotected and labelled in a ‘one pot’ reaction, yielding a ^99mTc‐BAT‐propylester of Ro‐15‐3890 (^99mTc‐techmazenil). Radio‐LC‐MS analysis of the isolated main peak showed the molecular ion mass (608.0618) of the expected ^99mTc‐techmazenil. The biodistribution of ^99mTc‐techmazenil was investigated in normal mice and indicated that the tracer is cleared from plasma mainly by the hepatobiliary system and shows a very low uptake in brain. In vitro binding studies on mice brain slices indicated that techmazenil does not bind to benzodiazepine receptors. Copyright © 2004 John Wiley & Sons, Ltd.     

Intracerebral microdialysis coupled to LC–MS/MS for the determination tramadol and its major pharmacologically active metabolite O‐desmethyltramadol in rat brain microdialysates

A rapid and sensitive method involving liquid chromatography electrospray tandem mass spectrometry (LC‐ESI‐MS/MS) coupled to an intracerebral microdialysis technique was developed for the determination and pharmacokinetic investigation of tramadol and its major active metabolite O ‐desmethyltramadol (ODT) in rat brain. The microdialysis samples were separated on a C18 column and eluted with a mobile phase of acetonitrile‐water‐formic acid (50:50:0.1; v/v/v ) at a flow rate of 0.3 mL/min. The ESI‐MS/MS spectra were performed in electrospray positive ion mode, and the analytes were detected by multiple reaction monitoring (MRM) of the transitions m/z [M + H]⁺ 264.3 → 58.2 for tramadol, m/z [M + H]⁺ 250.3 → 58.3 for ODT, and m/z [M + H]⁺ 379.4 → 264.0 for ambroxol (internal standard; IS). The total run time was 4.0 min. A lower limit of quantitation (LLOQ) was achieved as 1 ng/mL for tramadol and 0.5 ng/mL for ODT, with excellent linearity over a concentration range of 1 ~ 500 ng/mL (r  > 0.99) for tramadol and 0.5 ~ 50 ng/mL for ODT (r  > 0.99), respectively. The proposed method was successfully applied to the pharmacokinetic studies of tramadol and ODT in rat brain. Copyright © 2017 John Wiley & Sons, Ltd.     

Synthesis of a stable isotopically labeled universal surrogate peptide for use as an internal standard in LC‐MS/MS bioanalysis of human IgG and Fc‐fusion protein drug candidates

The synthesis of a 16‐residue, stable isotopically labeled peptide is described for use as a LC‐MS/MS (Liquid chromatography‐mass spectrometry/mass spectrometry) internal standard in bioanalytical studies. This peptide serves as a single universal surrogate peptide capable of quantifying a wide variety of immunoglobulin G and Fc‐fusion protein drug candidates in animal species used in pre‐clinical drug development studies. An efficient synthesis approach for this peptide was developed using microwave‐assisted solid phase peptide synthesis (SPPS) techniques, which included the use of a pseudoproline dipeptide derivative. The corresponding conventional room temperature SPPS was unsuccessful and gave only mixtures of truncated products. Stable‐labeled leucine was incorporated as a single residue via manual coupling of commercially available Fmoc‐[¹³C₆, ¹⁵N]‐l ‐leucine onto an 11‐unit segment followed by automated microwave‐assisted elaboration of the final four residues. Using this approach, the desired labeled peptide was prepared in high purity and in sufficient quantities for long‐term supplies as a bioanalytical internal standard. The results strongly demonstrate the importance of utilizing both microwave‐assisted peptide synthesis and pseudoproline dipeptide techniques to allow the preparation of labeled peptides with highly lipophilic and sterically hindered side‐chains.