UHPLC‐MS/MS and UHPLC‐HRMS identification of zolpidem and zopiclone main urinary metabolites and method development for their toxicological determination

Zolpidem and zopiclone (Z‐compounds) are non‐benzodiazepine hypnotics of new generation that can be used in drug‐facilitated sexual assault (DFSA). Their determination in biological fluids, mainly urine, is of primary importance; nevertheless, although they are excreted almost entirely as metabolites, available methods deal mainly with the determination of the unmetabolized drug. This paper describes a method for the determination in urine of Z‐compounds and their metabolites by ultra‐high‐pressure liquid chromatography/tandem mass spectrometry (UHPLC‐MS/MS) and UHPLC coupled with high resolution/high accuracy Orbitrap® mass spectrometry (UHPLC‐HRMS). The metabolic profile was studied on real samples collected from subjects in therapy with zolpidem or zopiclone; the main urinary metabolites were identified and their MS behaviour studied by MS/MS and HRMS. Two carboxy‐ and three hydroxy‐ metabolites, that could be also detected by gas chromatography/mass spectrometry (GC‐MS) as trimethylsylyl derivatives, have been identified for zolpidem. Also, at least one dihydroxilated metabolite was detected. As for zopiclone, the two main metabolites detected were N‐demethyl and N‐oxide zopiclone. For both substances, the unmetabolized compounds were excreted in low amounts in urine. In consideration of these data, a UHPLC‐MS/MS method for the determination of Z‐compounds and their main metabolites after isotopic dilution with deuterated analogues of zolpidem and zopiclone and direct injection of urine samples was set up. The proposed UHPLC‐MS/MS method appears to be practically applicable for the analysis of urine samples in analytical and forensic toxicology cases, as well as in cases of suspected DFSA. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of a selection of synthetic cannabinoids and metabolites in urine by UHPSFC‐MS/MS and by UHPLC‐MS/MS

Two different analytical techniques, ultra‐high performance supercritical fluid chromatography‐tandem mass spectrometry (UHPSFC‐MS/MS) and reversed phase ultra‐high performance liquid chromatography‐tandem mass spectrometry (UHPLC‐MS/MS), were used for the determination of two synthetic cannabinoids and eleven metabolites in urine; AM‐2201 N‐4‐OH‐pentyl, AM‐2233, JWH‐018 N‐5‐OH‐pentyl, JWH‐018 N‐pentanoic acid, JWH‐073 N‐4‐OH‐butyl, JWH‐073 N‐butanoic acid, JWH‐122 N‐5‐OH‐pentyl, MAM‐2201, MAM‐2201 N‐4‐OH‐pentyl, RCS‐4 N‐5‐OH‐pentyl, UR‐144 degradant N‐pentanoic acid, UR‐144 N‐4‐OH‐pentyl, and UR‐144 N‐pentanoic acid. Sample preparation included a liquid‐liquid extraction after deconjugation with ß‐glucuronidase. The UHPSFC‐MS/MS method used an Acquity UPC^{2 TM} BEH column with a mobile phase consisting of CO₂ and 0.3% ammonia in methanol, while the UHPLC‐MS/MS method used an Acquity UPLC® BEH C₁₈ column with a mobile phase consisting of 5 mM ammonium formate (pH 10.2) and methanol. MS/MS detection was performed with positive electrospray ionization and two multiple reaction monitoring transitions. Deuterated internal standards were used for six of the compounds. Limits of quantification (LOQs) were between 0.04 and 0.4 µg/L. Between‐day relative standard deviations at concentrations ≥ LOQ were ≤20%, with biases within ±19%. Recoveries ranged from 40 to 90%. Corrected matrix effects were within 100 ± 10%, except for MAM‐2201 with UHPSFC‐MS/MS, and for UR‐144 N‐pentanoic acid and MAM‐2201 N‐4‐OH‐pentyl with UHPLC‐MS/MS. Elution order obtained by UHPSFC‐MS/MS was almost opposite to that obtained by UHPLC‐MS/MS, making this instrument setup an interesting combination for screening and confirmation analyses in forensic cases. The UHPLC‐MS/MS method has, since August 2014, been successfully used for confirmation of synthetic cannabinoids in urine samples revealing a positive immunoassay screening result. Copyright © 2015 John Wiley & Sons, Ltd.     

Screening method for stimulants in urine by UHPLC‐MS/MS: identification of isomeric compounds

A fast screening method for the detection of more than 60 stimulants in urine was developed. The method consisted of a dilution of the urine (1:5 v/v) and analysis by ultra high performance liquid chromatography coupled to tandem mass spectrometry, using a C18 column (1.7 µm particle size), a mobile phase containing deionized water and acetonitrile with formic acid, and gradient elution. The chromatographic run time was 5 min. The detection was performed in positive mode electrospray ionization, monitoring one or two specific ion transitions for each analyte. Appropriate repeatability was obtained, with relative standard deviation (RSD) values below 25% for most of the analytes. Regarding intermediate precision, estimated during routine work, higher RSDs were obtained, probably due to between‐day differences in the status of the mass spectrometer and in the chromatographic system. Matrix effect ranged from 60 to 255% with RSD lower than 35% for the majority of compounds. Despite the matrix effect observed, the signal/noise ratio of the analytes spiked at 50 ng/mL was greater than three in all tested samples, allowing a correct detection of all substances at the minimum required performance levels required by the World Anti‐Doping Agency and demonstrating the suitability of the method. The method was tested in administration study samples and satisfactorily in operation for more than one year with routine doping samples. The presence of isomeric stimulants with closely similar chromatographic and/or mass spectrometric properties did not allow the unequivocal identification of these compounds after the first analysis. Different possibilities for separation and identification of isomeric compounds are presented. Copyright © 2015 John Wiley & Sons, Ltd.     

Targeted analysis of 116 drugs in hair by UHPLC‐MS/MS and its application to forensic cases

A multi‐target method that can detect a broad range of drugs in human hair, such as hypnotics, anxiolytics, analgesics, benzodiazepines, antihistamines, antidepressants, antipsychotics, and anticonvulsants, was developed based on ultra‐high‐performance liquid chromatography–tandem mass spectrometry (UHPLC‐MS/MS). The drugs were extracted from 10 mg of washed hair by incubation for 18 h in a 25:25:50 (v/v/v ) mixture of methanol/acetonitrile/2 mM ammonium formate (8% acetonitrile, pH 5.3). For 51% of the basic drugs, the lower limits of quantification (LLOQs) were in the range of 0.05–0.5 pg/mg, and the majority (98%) were ≤ 5 pg/mg. Linearity ranged from LLOQs to 100–500 pg/mg for all the basic drugs. For acid and neutral drugs, the LLOQs ranged from 0.4 to 500 pg/mg, and linearity ranged from LLOQs to 80–40 000 pg/mg. According to published reports on concentrations attained in single dose control studies, the present method is sensitive enough to detect single‐dose drug exposure for many of the drugs. The accuracy was within 75–125% for the majority of drugs. Good precision was observed (relative standard deviations [RSD%] < 25%) for most of the compounds, including the prepared quality control (QC) hair samples. The method was applied to forensic cases and concentrations of rarely reported drugs in hair in 25 post‐mortem forensic cases were presented. Hair concentrations of amisulpride, gabapentin, mianserin, mepyramine, orphenadrine, and xylometazoline have not been previously reported. Copyright © 2016 John Wiley & Sons, Ltd.     

UHPLC–MS/MS quantitation of porphyroxine in opium and application of porphyroxine‐acetylated products as signature markers for heroin

Porphyroxine, a trace alkaloid in opium, was identified in the early 1800s and isolated/characterized in the 1960s. Recently, two significant porphyroxine‐related byproducts found in the acidic and neutral extracts of illicit heroin were characterized by this laboratory as the N‐acetyl‐O¹⁴‐desmethyl‐epi‐porphyroxine ( B ) and N,O⁸‐diacetyl‐O¹⁴‐desmethyl‐epi‐porphyroxine ( C ). The prevalence of the B and C compounds has been consistent in the following order of abundance for the thousands of authentic heroin samples analyzed: Southwest Asia (SWA) > South America (SA) > Southeast Asia (SEA) > Mexico (MEX). In this research, a rapid and efficient ultra‐high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed to determine the content of porphyroxine and five primary alkaloids (morphine, codeine, thebaine, noscapine, and papaverine) in opium after extraction with methanol/water (50/50). The method was validated in terms of linearity, accuracy, recovery, and precision for porphyroxine. The limit of quantitation (LOQ) for porphyroxine was 2.5 ng/mL. The developed method was successfully applied to a total of 114 authentic opium samples from the major poppy‐growing regions. The amount of porphyroxine was determined at the level of part per thousand (‰) and the relative concentrations to morphine were in the range of 1x10^?4 and 1x10^?2 with an order of SWA > SEA, SA > MEX for its average abundance, which is consistent with the order of the average abundance of its acetylated products ( B , C ) in illicit heroin. This study reveals the significance of porphyroxine and its acylated compounds in classifying heroin and opium samples to major geographical regions of production.     

Development of a multi‐residue high‐throughput UHPLC–MS/MS method for routine monitoring of SARM compounds in equine and bovine blood

Selective androgen receptor modulators (SARMs) are a group of anabolic enhancer drugs posing threats to the integrity of animal sports and the safety of animal‐derived foods. The current research describes for the first time the development of a semi‐quantitative assay for the monitoring of SARM family compounds in blood and establishes the relative stability of these analytes under various storage conditions prior to analysis. The presented screening method validation was performed in line with current EU legislation for the inspection of livestock and produce of animal origin, with detection capability (CCβ) values determined at 0.5 ng/mL (Ly2452473), 1 ng/mL (AC‐262536 and PF‐06260414), 2 ng/mL (bicalutamide, GLPG0492, LGD‐2226, ostarine, S‐1, S‐6, and S‐23), and 5 ng/mL (andarine, BMS‐564929, LGD‐4033, RAD140, and S‐9), respectively. The applicability of the developed assay was demonstrated through the analysis of blood samples from racehorses and cattle. The developed method presents a high‐throughput cost‐effective tool for the routine screening for a range of SARM compounds in sport and livestock animals.     

Oral absorption,distribution, metabolism,and excretion of icaritin in rats by Q‐TOF and UHPLC–MS/MS

Icaritin (ICT) displays numerous pharmacological activities for the treatment of various cancers, osteoporosis, inflammation, and angiocardiopathy. The absorption, distribution, metabolism, and excretion of ICT still remain unknown. ICT was administered to rats at 2 mg/kg for intravenous injection or 40 mg/kg for oral route. Major metabolite of ICT was identified using quadrupole time‐of‐flight (Q‐TOF), and ICT and its major metabolite were quantified in plasma, tissues, urine, faeces, and bile by ultra‐high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS). A total of 24 metabolites of ICT in plasma were identified and mono C‐7 glucuronide glucuronidated icaritin (GICT) was the major metabolite of ICT after oral administration. The absolute bioavailability of ICT was 4.33% although ICT was rapidly absorbed into the blood. For oral administration, concentrations of GICT at various time points was 6.38–8.81‐fold higher than those of ICT, and the area under the curve (AUC) of GICT was about 8‐fold higher than that of ICT, while AUC values of ICT and GICT were almost equal for intravenous injection. Approximately 65.7% ICT and 42.7% GICT were distributed in liver and kidney, respectively. Unabsorbed ICT was mainly excreted as the parent form in faeces with at least 60% of administered dose during 24 h, whereas absorbed ICT was predominantly excreted as GICT from urine with 2.74% of administered dose accounting for 63.28% of absorbed drug. ICT was rapidly absorbed into the blood although a large amount of ICT remained unabsorbed, and then rapidly and mainly metabolized to GICT. ICT mainly distributed in liver, while GICT predominantly distributed in kidney. Absorbed ICT and GICT were predominantly excreted via urine, and unabsorbed ICT was mainly excreted as the parent form in faeces. Copyright © 2017 John Wiley & Sons, Ltd.     

Targeted and non‐targeted drug screening in whole blood by UHPLC‐TOF‐MS with data‐independent acquisition

High‐resolution mass spectrometry (HRMS) is widely used for the drug screening of biological samples in clinical and forensic laboratories. With the continuous addition of new psychoactive substances (NPS), keeping such methods updated is challenging. HRMS allows for combined targeted and non‐targeted screening. First, peaks are identified by software algorithms, and identifications are based on reference standard data. Attempts are made to identify the remaining unknown peaks with in silico and literature data. However, several thousand peaks remain where most are unidentifiable or uninteresting in drug screening. The aims of the study were to apply a combined targeted and non‐targeted screening approach to authentic driving‐under‐the‐influence‐of‐drugs (DUID) samples (n = 44) and further validate the approach using whole‐blood samples spiked with 11 low‐dose synthetic benzodiazepine analogues (SBAs). Analytical data were acquired using ultra‐high‐performance liquid chromatography coupled with a time‐of‐flight mass spectrometer (UHPLC‐TOF‐MS) with data‐independent acquisition (DIA). We present a combined targeted and non‐targeted screening, where peak deconvolution and filtering reduced the number of peaks to inspect by three orders of magnitude, down to four peaks per DUID sample. The screening allowed for tentative identification of metabolites and drugs not included in the initial screening; 3 drugs and 14 metabolites were tentatively identified in the authentic DUID samples. Running targeted‐screening true‐positive identifications through the filters retained 73% of identifications. In the non‐targeted screening, nine of the spiked SBAs were identified in the concentration range of 0.005–0.1 mg/kg, of which three were tentatively identified at concentrations below those reported in the literature. Copyright © 2016 John Wiley & Sons, Ltd.     

Patterns of drug abuse among drug users with regular and irregular attendance for treatment as detected by comprehensive UHPLC‐HR‐TOF‐MS

The most severe consequences of drug abuse include infectious diseases, overdoses, and drug‐related deaths. As the range of toxicologically relevant compounds is continually changing due to the emergence of new psychoactive substances (NPS), laboratories are encountering analytical challenges. Current immunoassays are insufficient for determining the whole range of the drugs abused, and a broad‐spectrum screening method is therefore needed. Here, the patterns of drug abuse in two groups of drug users were studied from urine samples using a comprehensive screening method based on high‐resolution time‐of‐flight mass spectrometry. The two groups comprised drug abusers undergoing opioid maintenance treatment (OMT) or drug withdrawal therapy and routinely visiting a rehabilitation clinic, and drug abusers with irregular attendance at a harm reduction unit (HRU) and suspected of potential NPS abuse. Polydrug abuse was observed in both groups, but was more pronounced among the HRU subjects with a mean number of concurrent drugs per sample of 3.9, whereas among the regularly treated subjects the corresponding number was 2.1. NPS and pregabalin were more frequent among HRU subjects, and their abuse was always related to drug co‐use. The most common drug combination for an HRU subject included amphetamine, cannabis, buprenorphine, benzodiazepine, and alpha‐pyrrolidinovalerophenone. A typical set of drugs for treated subjects was buprenorphine, benzodiazepine, and occasionally amphetamine. Abuse of several concurrent drugs poses a higher risk of drug intoxication and a threat of premature termination of OMT. Since the subjects attending treatment used fewer concurrent drugs, this treatment could be valuable in reducing polydrug abuse. Copyright © 2015 John Wiley & Sons, Ltd.     

Ultra‐fast LC–MS/MS in therapeutic drug monitoring: Quantification of clozapine and norclozapine in human plasma

A novel approach to high‐throughput, targeted liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis has been developed. A single chromatographic system can be used for the analysis of a range of 20 drugs and metabolites with a total analysis time of 36 s (one 96‐well plate of prepared samples per hour). To demonstrate the applicability of this approach to quantitative analysis, a method has been validated for the therapeutic drug monitoring of clozapine and norclozapine following automated extraction from human plasma. Chromatographic retention times were 11.4 and 12.4 s for norclozapine and clozapine, respectively (for both analytes the chromatographic peak width was less than 1 s). Comparison with a conventional LC–MS/MS method (5 min analysis time) showed excellent agreement. This new approach offers analysis times more akin to flow‐injection analysis, but is likely to be more widely applicable because of chromatographic resolution from residual matrix components and isobaric interferences.     

Determination of 21 drugs in oral fluid using fully automated supported liquid extraction and UHPLC‐MS/MS

Collection of oral fluid (OF) is easy and non‐invasive compared to the collection of urine and blood, and interest in OF for drug screening and diagnostic purposes is increasing. A high‐throughput ultra‐high‐performance liquid chromatography‐tandem mass spectrometry method for determination of 21 drugs in OF using fully automated 96‐well plate supported liquid extraction for sample preparation is presented. The method contains a selection of classic drugs of abuse, including amphetamines, cocaine, cannabis, opioids, and benzodiazepines. The method was fully validated for 200 μL OF/buffer mix using an Intercept OF sampling kit; validation included linearity, sensitivity, precision, accuracy, extraction recovery, matrix effects, stability, and carry‐over. Inter‐assay precision (RSD) and accuracy (relative error) were <15% and 13 to 5%, respectively, for all compounds at concentrations equal to or higher than the lower limit of quantification. Extraction recoveries were between 58 and 76% (RSD < 8%), except for tetrahydrocannabinol and three 7‐amino benzodiazepine metabolites with recoveries between 23 and 33% (RSD between 51 and 52 % and 11 and 25%, respectively). Ion enhancement or ion suppression effects were observed for a few compounds; however, to a large degree they were compensated for by the internal standards used. Deuterium‐labelled and ¹³C‐labelled internal standards were used for 8 and 11 of the compounds, respectively. In a comparison between Intercept and Quantisal OF kits, better recoveries and fewer matrix effects were observed for some compounds using Quantisal. The method is sensitive and robust for its purposes and has been used successfully since February 2015 for analysis of Intercept OF samples from 2600 cases in a 12‐month period. Copyright © 2016 John Wiley & Sons, Ltd.     

Surveillance of drug abuse in Hong Kong by hair analysis using LC‐MS/MS

The aim of this study is to reveal the habits of drug abusers in hair samples from drug rehabilitation units in Hong Kong. With the application of liquid chromatography–tandem mass spectrometry (LC–MS/MS) technology, a total of 1771 hair samples were analyzed during the period of hair testing service (January 2012 to March 2016) provided to 14 drug rehabilitation units including non‐governmental organizations (NGOs), rehabilitation centers, and medical clinics. Hair samples were analyzed for abused drugs and their metabolites simultaneously, including ketamine, norketamine, cocaine, benzoylecgonine, cocaethylene, norcocaine, codeine, MDMA, MDA, MDEA, amphetamine, methamphetamine, morphine, 6‐acetylmorphine, phencyclidine, and methadone. The results showed that ketamine (77.2%), cocaine (21.3%), and methamphetamine (16.5%) were the frequently detected drugs among those drug abusers, which is consistent with the reported data. In addition, the usage of multiple drugs was also observed in the hair samples. About 29% of drug‐positive samples were detected with multiple drug use. Our studies prove that our locally developed hair drug‐testing method and service can be a valid tool to monitor the use of abused drugs, and which could facilitate rehabilitation program management.     

Screening,quantification, and confirmation of synthetic cannabinoid metabolites in urine by UHPLC–QTOF–MS

Synthetic cannabinoids are one of the most significant groups within the category new psychoactive substances (NPS) and in recent years new compounds have continuously been introduced to the market of recreational drugs. A sensitive and quantitative screening method in urine with metabolites of frequently seized compounds in Norway (AB‐FUBINACA, AB‐PINACA, AB‐CHMINACA, AM‐2201, AKB48, 5F‐AKB48, BB‐22, JWH‐018, JWH‐073, JWH‐081, JWH‐122, JWH‐203, JWH‐250, PB‐22, 5F‐PB‐22, RCS‐4, THJ‐2201, and UR‐144) using ultra‐high pressure liquid chromatography–quadrupole time of flight–mass spectrometry (UHPLC–QTOF–MS) has been developed. The samples were treated with ß‐glucuronidase prior to extraction and solid‐phase extraction was used. Liquid handling was automated using a robot. Chromatographic separation was achieved using a C18‐column and a gradient of water and acetonitrile, both with 0.1% formic acid. Each sample was initially screened for identification and quantification followed by a second injection for confirmation. The concentrations by which the compounds could be confirmed varied between 0.1 and 12 ng/mL. Overall the validation showed that the method fulfilled the set criteria and requirements for matrix effect, extraction recovery, linearity, precision, accuracy, specificity, and stability. One thousand urine samples from subjects in drug withdrawal programs were analyzed using the presented method. The metabolite AB‐FUBINACA M3, hydroxylated metabolite of 5F‐AKB48, hydroxylated metabolite of AKB48, AKB48 N‐pentanoic acid, 5F‐PB‐22 3‐carboxyindole, BB‐22 3‐carboxyindole, JWH‐018 N‐(5‐hydroxypentyl), JWH‐018 N‐pentanoic acid, and JWH‐073 N‐butanoic acid were quantified and confirmed in 2.3% of the samples. The method was proven to be sensitive, selective and robust for routine use for the investigated metabolites.     

The preparation of genistein and LC‐MS/MS on‐line analysis

Genistein, a promising agent for cancer chemoprevention or treatment, can be obtained from soybean seeds, via a complex isolation procedure. The objective of this study was to investigate whether natural and high purity genistein could be acquired from Sophora japonica L. via a simple approach. High‐performance liquid chromatography electrospray ionization combined with tandem mass spectrometry (LC‐ESI‐MS/MS) was used to monitor the whole process of genistein preparation. The results showed there were at least 13 types of flavonoid in the crude extracts from sophora fruits with sophoricoside being the predominant component. The products obtained from our preparation were unambiguously identified as genistein based on molecular ion [M+H]⁺, UV spectra, retention time, IR spectra, ¹HNMR, ¹⁴CNMR spectra, and tandem mass spectrometric analysis. It demonstrated that 99.6% (w/w) genistein could be obtained via our preparation protocol. Drug Dev. Res. 61: 6–12, 2004. © 2004 Wiley‐Liss, Inc.     

Metabolite fingerprinting of Camptotheca acuminata and the HPLC–ESI-MS/MS analysis of camptothecin and related alkaloids

The major phytochemical constituents, namely, alkaloids, flavonoids and ellagic acid derivatives, of leaves of Camptotheca acuminata were identified using high performance liquid chromatography (HPLC) coupled with electrospray mass spectrometry (ESI-MS) in extracts of plants cultivated in Italy and collected at different growth stages. Alkaloids related to camptothecin were identified and quantified by HPLC coupled with ESI-tandem mass spectrometry (MS/MS) employing, respectively, an ion trap and a triple quadrupole mass analyser. The fragmentation patterns of alkaloids related to camptothecin were analysed and a specific Multiple Reaction Monitoring HPLC–MS/MS method was developed for the quantitative determination of these constituents. The described method provides high sensitivity and specificity for the characterisation and quantitative determination of the alkaloids in C. acuminata.     

Segmental hair analysis for differentiation of tilidine intake from external contamination using LC‐ESI‐MS/MS and MALDI‐MS/MS imaging

Segmental hair analysis has been used for monitoring changes of consumption habit of drugs. Contamination from the environment or sweat might cause interpretative problems. For this reason, hair analysis results were compared in hair samples taken 24 h and 30 days after a single tilidine dose. The 24‐h hair samples already showed high concentrations of tilidine and nortilidine. Analysis of wash water from sample preparation confirmed external contamination by sweat as reason. The 30‐day hair samples were still positive for tilidine in all segments. Negative wash‐water analysis proved incorporation from sweat into the hair matrix. Interpretation of a forensic case was requested where two children had been administered tilidine by their nanny and tilidine/nortilidine had been detected in all hair segments, possibly indicating multiple applications. Taking into consideration the results of the present study and of MALDI‐MS imaging, a single application as cause for analytical results could no longer be excluded. Interpretation of consumption behaviour of tilidine based on segmental hair analysis has to be done with caution, even after typical wash procedures during sample preparation. External sweat contamination followed by incorporation into the hair matrix can mimic chronic intake. For assessment of external contamination, hair samples should not only be collected several weeks but also one to a few days after intake. MALDI‐MS imaging of single hair can be a complementary tool for interpretation. Limitations for interpretation of segmental hair analysis shown here might also be applicable to drugs with comparable physicochemical and pharmacokinetic properties. Copyright © 2014 John Wiley & Sons, Ltd.     

Monitoring drug residues in donor blood/plasma samples using LC‐(MS)/MS – a pilot study

Quality assurance of pharmaceutical products is of particular importance and thoroughly controlled. Among these, the preparation of human plasma follows strict guidelines from the point of donor selection to product processing. While various precautions particularly concerning antiviral treatment as well as quality assessment are standard procedure, tests for drug residues are rarely, if at all, conducted with fresh frozen plasma products. With the constantly increasing sensitivity and specificity of modern analytical instruments, the detection of trace amounts of therapeutics in plasma is feasible and can be applied to blood products where considered appropriate. To estimate the prevalence of a selection of commonly prescribed and over‐the‐counter drugs (including diuretics, beta‐receptor blocking agents, contraceptives, β₂‐agonists, antibiotics, antidepressants, analgesics, opioids, glucocorticosteroids, benzodiazepines, stimulants, and oral anti‐diabetics) as well as cannabinoids in human donor plasma, a total of 100 specimens (61 female, 39 male) collected at the German Red Cross Organization in 2012 was subjected to an established analytical approach. The methodology was based on protein precipitation followed by liquid chromatographic‐high resolution/high accuracy mass spectrometric analysis. Following initial test results, confirmatory analyses were conducted with respective reference substances employing a conventional liquid chromatography‐triple‐quadrupole mass spectrometer (LC‐MS/MS) apparatus. Out of one hundred samples, five were found to contain diuretics (four hydrochlorothiazide and one torasemide), five contained beta‐receptor blocking agents (four bisoprolol and one metoprolol), one was found with residues of pseudoephedrine (stimulant) and one with drosperinone (contraceptive). Overall, 12% of samples yielded detectable amounts of drug residues at concentrations estimated to levels common to individuals under therapeutic treatment. In addition, six aliquots of different lots of commercially available plasma preparations with solvent‐detergent processing were tested. Here, no drug residues of the targeted therapeutics were detected. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and optimization of on‐line 2‐dimensional chromatographic approaches for eliminating matrix effects and improving bioanalysis of peptides in human plasma using UHPLC‐MS/MS

Online 2‐dimensional chromatographic approaches for eliminating matrix effects and optimizing bioanalysis of peptides using ultra‐high performance liquid chromatography‐tandem mass spectrometry (UHPLC‐MS/MS) were studied. Three therapeutic peptides (octreotide, desmopressin, and vasopressin) were selected as model analytes. Human plasma was precipitated with acetonitrile; peptides were analyzed on C₈, C₁₈, Phenyl and HILIC ACQUITY UPLC columns. For simpler online clean‐up applications, a C₁₈ pre‐column was coupled to the analytical column via a switching valve. For more complex heart‐cutting applications, two analytical columns were used with optional online dilution to refocus the analyte peaks prior to the second dimension separation. This allows the use of MS incompatible mobile phases, such as TFA, in the first dimension separation. Online clean‐up effectiveness was investigated by monitoring phospholipids. Flushing direction, mobile phase composition, flow rate and transfer window were evaluated. Phospholipids were readily retained on reversed‐phase columns, and the peptides were reproducibly transferred, individually or as a group, to the second column using appropriate transfer windows. The best peak shapes were obtained when the second dimension column was more retentive (e.g. C₁₈ vs. C₈). However, C₈ to HILIC gave broad unresolved peaks due to mobile phase mismatch. Trapped phospholipids were efficiently removed from either guard columns or first dimensional columns by forward‐ or back‐flushing at high flows; however, back‐flushing was more efficient with lower flow rates on larger columns. Copyright © 2013 John Wiley & Sons, Ltd.