Liquid chromatography–tandem mass spectrometry analysis of cocaine and its metabolites from blood, amniotic fluid, placental and fetal tissues: study of the metabolism and distribution of cocaine in pregnant rats

The ability to simultaneously quantitate cocaine and its 12 metabolites from pregnant rat blood, amniotic fluid, placental and fetal tissue homogenates aids in elucidating the metabolism and distribution of cocaine. An efficient extraction method was developed to simultaneously recover these 13 components using underivatized silica solid-phase extraction (SPE) cartridges. The overall recoveries for cocaine and its metabolites were studied from pregnant rat blood (47–100%), amniotic fluid (61–100%), placental homogenate (31–83%), and fetal homogenate (39–87%). Extraction of the samples using silica is not classical SPE, but rather allows for the concentration of the sample into a small volume prior to injection and the removal of the proteins due to their strong interaction with the active silica surface. A positive ion mode electrospray ionization liquid chromatography–tandem mass spectrometry (LC–MS–MS) method was used and validated to simultaneously quantitate cocaine and 12 metabolites from these four biological matrices. A gradient elution method with a Zorbax XDB C₈ reversed-phase column was used to separate the components. Multiple reaction monitoring (MRM) of a product ion arising from the corresponding precursor ion was used in order to enhance the selectivity and sensitivity of the method. Low background noise was observed from the complex biological matrices due to efficient SPE and the selectivity of the MRM mode. Linear calibration curves were generated from 0.01 to 2.50 ppm. The method also showed high intra-day (n=3) and inter-day (n=9) precision (% RSD) and accuracy (% error) for all components. The limits of detection (LODs) for the method ranged from 0.15 to 10 ppb. The LODs of cocaine and its major metabolites were less than 1 ppb from all four biological matrices. This method was applied to the study of the metabolism and distribution of cocaine in pregnant rats following intravenous infusion to a steady state plasma drug concentration. The following results were observed in the pregnant rat study: (1) the observations correlated strongly with the previous literature data on cocaine metabolism and distribution, (2) cocaine and norcocaine accumulated in the placenta, (3) arylhydroxylation of cocaine was a major metabolic pathway, (4) para-arylhydroxylation of cocaine was favored over meta-arylhydroxylation in rats and (5) accumulation of cocaine and its major metabolites was observed in the amniotic fluid.     

Development and validation of a LC/MS/MS method for simultaneous quantification of oxcarbazepine and its main metabolites in human serum

A fast, sensitive and specific LC/MS/MS method for the simultaneous analysis of oxcarbazepine (OXC), 10-hydroxycarbazepine (MHD) and trans-diol-carbazepine (DHD), in human serum, has been developed and validated. Serum drugs were extracted by C8 solid-phase cartridges (SPE) and separated in less than 3 min on a C18 reverse-phase column using an isocratic elution. A tandem mass spectrometer, as detector, was used for quantitative analysis in positive mode by a multiple reaction monitoring. Calibration curves, obtained on two ranges of concentration (0.78-50 mg/L for MHD and 0.078-5.0 mg/L for OXC and DHD), showed correlation coefficients (r) better than 0.997. Within day and between days quality controls imprecision, as CV%, ranged from 0.3 to 4.6% and from 1.9 to 5.8%, respectively. Cyheptamide (CYE) was used as internal standard. No detectable carry-over and no relevant cross-talk and matrix effect occurred. Samples from 24 treated patients were analysed and drug serum concentrations obtained by this method are in agreement with those of other methods and also are well correlated (r=0.88) in comparison to our routine HPLC-UV method. Based on the analytical results and short run time, the method is suitable to support routine analysis of therapeutic drugs monitoring from human serum of treated patients or for pharmacokinetic studies.     

Development and validation of a gas chromatography-mass spectrometry method for the simultaneous determination of buprenorphine, flunitrazepam and their metabolites in rat plasma: application to the pharmacokinetic study

Buprenorphine (BUP), a synthetic opioid analgesic, is frequently abused alone, and in association with benzodiazepines. Fatalities involving buprenorphine alone seem very unusual while its association with benzodiazepines, such as flunitrazepam (FNZ), has been reported to result in severe respiratory depression and death. The quantitative relationship between these drugs remain, however, uncertain. Our objective was to develop an analytical method that could be used as a means to study and explore, in animals, the toxicity and pharmacological interaction mechanisms between buprenorphine, flunitrazepam and their active metabolites. A procedure based on gas chromatography-mass spectrometry (GC-MS) is described for the simultaneous analysis of buprenorphine, norbuprenorphine (NBUP), flunitrazepam, N-desmethylflunitrazepam (N-DMFNZ) and 7-aminoflunitrazepam (7-AFNZ) in rat plasma. The method was set up and adapted for the analysis of small plasma samples taken from rats. Plasma samples were extracted by liquid-liquid extraction using Toxi-tubes A. Extracted compounds were derivatized with N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA), using trimethylchlorosilane (TMCS) as a catalyst. They were then separated by GC on a crosslinked 5% phenyl-methylpolysiloxane analytical column and determined by a quadrupole mass spectrometer detector operated under selected ion monitoring mode. Excellent linearity was found between 0.125 and 25 ng/microl plasma for BUP, 0.125 and 12.5 ng/microl for NBUP and N-DMFNZ, 0.125 and 5 ng/microl for FNZ, and between 0.025 and 50 ng/microl for 7-AFNZ. The limit of quantification was 0.025 ng/microl plasma for 7-AFNZ and 0.125 ng/microl for the four other compounds. A good reproducibility (intra-assay CV=0.32-11.69%; inter-assay CV=0.63-9.55%) and accuracy (intra-assay error=2.58-12.73%; inter-assay error=0.83-11.07%) were attained. Recoveries were 71, 67 and 81%, for BUP, FNZ and N-DMFNZ, respectively, and 51% for NBUP and 7-AFNZ, with CV ranging from 5.4 to 13.9%, and were concentration-independent. The GC-MS method was successfully applied to the pharmacokinetic study of BUP, NBUP, FNZ, DMFNZ and 7-AFNZ in rats, after administration of BUP and FNZ.     

Liquid chromatography/tandem mass spectrometry assay for the simultaneous determination of cefoperazone and sulbactam in plasma and its application to a pharmacokinetic study

A rapid and highly sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for simultaneous determination of cefoperazone sodium and sulbactam sodium in human plasma was developed. The analytes and internal standard (IS), cefuroxime sodium, were extracted from human plasma via liquid-liquid extraction with ethyl acetate and separated on a Waters Xterra C18 column within 3.5 min. Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique, operating in selected reaction monitoring (SRM) and negative ion mode. The precursor to product ion transitions monitored for cefoperazone, sulbactam and IS were m/z 644.1→528.0, 232.1→140.0, and 423.0→362.0, respectively. The assay was validated in the linear range of 0.1-20 μg/mL for cefoperazone and 0.02-4 μg/mL for sulbactam. The intra- and inter-day precisions (CV%) were within 8.39% for each analyte. The recoveries were greater than 87.3% for cefoperazone and 87.2% for sulbactam. Each analyte was found to be stable during all sample storage, preparation and analytical procedures. The method was successfully applied in a pharmacokinetic study of Sulperazon injection in six hospital-acquired pneumonia (HAP) patients.     

Development and validation of a rapid and sensitive assay for simultaneous quantification of midazolam, 1′-hydroxymidazolam,and 4-hydroxymidazolam by liquid chromatography coupled to tandem mass-spectrometry

Midazolam is an ultra short acting benzodiazepine derivative and a specific probe for phenotyping cytochrome P450 (P450) 3A4/5 activity. A rapid, sensitive, and selective LC–MS/MS method was developed for simultaneous quantitation of midazolam and its metabolites (1′-hydroxymidazolam and 4-hydroxymidazolam). Deuterated (D₅) analog of midazolam was utilized as an internal standard. Sample preparation either from human plasma (100 μL) or liver microsomal incubations involved a simple protein precipitation using acetonitrile (900 μL) with an average recovery of >90% for all compounds. The chromatographic separation was achieved using Zorbax-SB Phenyl, Rapid Resolution HT (2.1 mm × 100 mm, 3.5 μm) and a gradient elution with 10 mM ammonium acetate in 10% methanol (A) and acetonitrile (B). The flow rate was 0.25 mL/min and total run time was 5.5 min. Calibration curves were linear over the concentration range of 0.100–250 ng/mL. The lower limit of quantitation (LLOQ) was 0.1 ng/mL for all three analytes. The accuracy and precision, estimated at LLOQ and three concentration levels of quality control samples in six replicates, were within 85–115%. In conclusion, a robust, simple and highly sensitive analytical method was developed and validated for the analysis of midazolam and its metabolites. This method is suitable for characterizing the P450 3A4/5 activity in vitro or in human pharmacokinetic studies allowing administration of smaller doses of midazolam.     

Development and validation of a liquid chromatography-tandem mass spectrometry method for the determination of phenylephrine in human plasma and its application to a pharmacokinetic study

A sensitive and reliable method was developed to quantitate phenylephrine in human plasma using liquid chromatography-electrospray tandem mass spectrometry. The assay was based on solid-phase extraction with C18 cartridges and hydrophilic interaction chromatography performed on a pentafluorophenylpropylsilica column (50 mm x 4 mm, 3 microm particles), the mobile phase consisted of methanol-10 mM ammonium acetate (90:10, v/v). Quantification was through positive-ion mode and selected reaction monitoring at m/z 168.1-->135.0 for phenylephrine and m/z 182.1-->135.0 for internal standard etilefrin, respectively. The lower limit of quantitation was 51 pg/ml using 0.25 ml of plasma and linearity was observed from 51 to 5500 pg/ml. Within-day and between-day precision expressed by relative standard deviation was less than 12% and inaccuracy did not exceed 8% at all levels. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Development and validation of a liquid chromatography/tandem mass spectrometry assay for the simultaneous determination of D-amphetamine and diphenhydramine in beagle dog plasma and its application to a pharmacokinetic study

A new drug, quick-acting anti-motion capsule (QAAMC) composed of d-amphetamine sulfate, dimenhydrinate and ginger extraction has been studied for anti-motion-sickness use. We have developed a sensitive, specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantitative determination of d-amphetamine and diphenhydramine, the main effective components of the QAAMC, using pseudoephedrine as the internal standard. The analytes and internal standard were isolated from 200 microL plasma samples by a simple liquid-liquid extraction (LLE). Reverse-phase HPLC separation was accomplished on a Zorbax SB-C18 column (100 mm x 3.0 mm, 3.5 microm) with a mobile phase composed of methanol-water-formic acid (65:35:0.5, v/v/v) at a flow rate of 0.2 mL/min. The method had a chromatographic total run time of 5 min. A Varian 1200 L electrospray tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 136.0-->91.0 (D-amphetamine), 256.0-->167.0 (diphenhydramine) and 166.1-->148.0 (IS) used for quantitation. The method was sensitive with a lower limit of quantitation (LLOQ) of 0.5 ng/mL for d-amphetamine and 1 ng/mL for diphenhydramine, with good linearity in the range 0.5-200 ng/mL for D-amphetamine and 1-500 ng/mL for diphenhydramine (r(2)> or =0.9990). All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. The method was successfully applied to pharmacokinetic study of the QAAMC in beagle dogs.     

Hydrophilic interaction liquid chromatography-tandem mass spectrometry for the determination of adefovir in human plasma and its application to a pharmacokinetic study

A selective, rapid and sensitive hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) method was developed for the first time to determine adefovir in human plasma and applied to a pharmacokinetic study. Plasma samples were prepared by protein precipitation with methanol followed by a further cleaning using dichloromethane. The chromatographic separation was carried out on an ACQUITY UPLC™ BEH HILIC column with the mobile phase of methanol–water–formic acid (85:15:0.2, v/v/v). The detection was performed on a triple-quadrupole tandem mass spectrometer with multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The method was rapid with a run time of 3 min per sample. The linear calibration curves were obtained in the concentration range of 1.02–102 ng/mL (r² ≥ 0.99) with the lower limit of quantification (LLOQ) of 1.02 ng/mL. The intra- and inter-day precision (relative standard deviation, R.S.D.) values were below 12% and the accuracy (relative error, R.E.) was from 0.6% to 3.2% at all quality control (QC) levels. The method was applicable to clinical pharmacokinetic study of adefovir in healthy volunteers after oral administration of adefovir dipivoxil tablet.     

Development and validation of a liquid chromatography/tandem mass spectrometry assay for the simultaneous determination of nateglinide, cilostazol and its active metabolite 3,4-dehydro-cilostazol in Wistar rat plasma and its application to pharmacokinetic study

Nateglinide (NTG), an insulin secretogogue, has been studied in rats for drug-drug interaction with cilostazol (CLZ), an antiplatelet agent commonly used in diabetics. We developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) based method that is capable of simultaneous monitoring plasma levels of nateglinide, cilostazol, and its active metabolite 3,4-dehydro-cilostazol (DCLZ). All analytes including the internal standard (Repaglinide) were chromatographed on reverse phase C(18) column (50 mm x 4.6mm i.d., 5 microm) using acetonitrile: 2mM ammonium acetate buffer, pH 3.4 (90:10, v/v) as mobile phase at a flow rate 0.4 ml/min in an isocratic mode. The detection of analyte was performed on LC-MS/MS system in the multiple reaction monitoring (MRM) mode. The quantitations for analytes were based on relative concentration. The method was validated over the concentration range of 20-2000 ng/ml and the lower limit of quantitation was 20 ng/ml. The recoveries from spiked control samples were >79% for all analytes and internal standard. Intra- and inter-day accuracy and precision of validated method were with in the acceptable limits of <15% at all concentration. The quantitation method was successfully applied for simultaneous estimation of NTG, CLZ and DCLZ in a pharmacokinetic drug-drug interaction study in Wistar rats.     

Optimization of lentiviral transduction parameters and its application for CRISPR-based secretome modification of human endometrial mesenchymal stem cells

Mesenchymal stem cells (MSCs) hold a great promise for successful development of regenerative medicine. Among the plenty of uncovered MSCs sources, desquamated endometrium collected from the menstrual blood probably remains the most accessible. Though numerous studies have been published on human endometrium-derived mesenchymal stem cells (hMESCs) properties in the past years, there are only a few data regarding their genetic modulation. Moreover, there is a lack of information about the fate of the transduced hMESCs. The present study aimed to optimize hMESCs transduction parameters and apply Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology for genome and secretome modification. The fate of hMESCs transduced either in presence of polybrene (Pb) or protamine sulfate (Ps) was assessed by alterations in CD expression profile, growth rate, cell size, migration capability, osteogenic, adipogenic, and decidual differential potentials. Here, we postulated that the use of Ps for hMESCs genetic manipulations is preferable, as it has no impact on the stem-cell properties, whereas Pb application is undesirable, as it induces cellular senescence. Plasminogen activator inhibitor-1 was selected for further targeted hMESCs genome and secretome modification using CRISPR/Cas9 systems. The obtained data provide optimized transduction scheme for hMESCs and verification of its effectiveness by successful hMESCs genome editing via CRISPR/Cas9 technology.     

Development and validation of a liquid chromatography-tandem mass spectrometry method for the determination of xanthinol in human plasma and its application in a bioequivalence study of xanthinol nicotinate tablets

A sensitive, rapid liquid chromatographic-electrospray ionization mass spectrometric method for the determination of xanthinol in human plasma was developed and validated. Xanthinol nicotinate in plasma (0.5mL) was pretreated with 20% trichloroacetic acid for protein precipitation. The samples were separated using a Lichrospher silica (5mum, 250mmx4.6mm i.d.). A mobile phase of methanol-water containing 0.1% formic acid (50: 50, v/v) was used isocratically eluting at a flow rate of 1mL/min. Xanthinol and its internal standard (IS), acyclovir, were measured by electrospray ion source in positive selected reaction monitoring mode. The method demonstrated that good linearity ranged from 10.27 to 1642.8ng/mL with r=0.9956. The limit of quantification for xanthinol in plasma was 10.27ng/mL with good accuracy and precision. The mean plasma extraction recovery of xanthinol was in the range of 90.9-100.2%. The intra- and inter-batch variability values were less than 4.8% and 7.9% (relative standard deviation, R.S.D.), respectively. The established method has been successfully applied to a bioequivalence study of two xanthinol nicotinate tablets for 20 healthy volunteers.     

Development and validation of a liquid chromatography/tandem mass spectrometry procedure for the quantification of sunitinib (SU11248) and its active metabolite, N-desethyl sunitinib (SU12662), in human plasma: application to an explorative study

A sensitive, precise and accurate quantitative liquid chromatography/tandem mass spectrometry (LC–MS/MS) method for the measurement of sunitinib (SU11248) and N-desethyl sunitinib (SU12662) in human plasma was developed and validated. All sample handling was done under strict light protection. The sample preparation method employed acetonitrile protein precipitation using d₅-SU11248 as an internal standard. The processed samples were chromatographed on a polymeric reversed-phase analytical column and analyzed by triple-quadrupole MS/MS in multiple reaction monitoring (MRM) mode using positive TurboIonSpray^® (TISP). The LC–MS/MS method described in this paper presents high absolute recovery (86.2% SU11248, 84.8% SU12662), high sensitivity (lower limit of quantitation of 0.06 ng/mL for both analytes), high inter-day precision (1.6–6.1% SU11248, 1.1–5.3% SU12662) and high analytical recovery (99.8–109.1% SU11248, 99.9–106.2% SU12662), as well as excellent linearity over the concentration range 0.060–100 ng/mL (r² > 0.999) with a short runtime of only 4.0 min. Results on the stability of SU11248 and SU12662 in human plasma are presented. During validation plasma from intensive care patients receiving many drugs were tested for interference and incurred samples were analyzed. The method met all criteria of the EMA and FDA guidelines during validation and was successfully applied to a pharmacokinetic study in healthy human volunteers.     

Development and validation of a high-throughput method for the quantitative analysis of d-amphetamine in rat blood using liquid chromatography/MS on a hybrid triple quadrupole-linear ion trap mass spectrometer and its application to a pharmacokinetic study

Amphetamines are a group of sympathomimetic drugs that exhibit strong central nervous system stimulant effects. d-Amphetamine ((+)-alpha-methylphenetylamine) is the parent drug in this class to which all others are structurally related. In drug discovery, d-amphetamine is extensively used either for the exploration of novel mechanisms involving the catecholaminergic system, or for the validation of new behavioural animal models. Due to this extensive use of d-amphetamine in drug research and its interest in toxicologic–forensic investigation, a specific and high-throughput method, with minimal sample preparation, is necessary for routine analysis of d-amphetamine in biological samples. We propose here a sensitive, specific and high-throughput bioanalytical method for the quantitative determination of d-amphetamine in rat blood using MS³ scan mode on a hybrid triple quadrupole-linear ion trap mass spectrometer (LC–MS/MS/MS). Blood samples, following dilution with water, were prepared by fully automated protein precipitation with acetonitrile containing an internal standard. The chromatographic separation was achieved on a Waters XTerra C18 column (2.1 mm × 30 mm, 3.5 μm) using gradient elution at a flow rate of 1.0 mL/min over a 2 min run time. An Applied Biosystems API4000 QTRAP™ mass spectrometer equipped with turbo ion-spray ionization source was operated simultaneously in MS³ scan mode for the d-amphetamine and in multiple reaction monitoring (MRM) for the internal standard. The MS/MS/MS ion transition monitored was m/z 136.1 → 119.1 → 91.1 for the quantitation of d-amphetamine and for the internal standard (rolipram) the MS/MS ion transition monitored was m/z 276.1 → 208.2. The linear dynamic range was established over the concentration range 0.5–1000 ng/mL (r² = 0.9991). The method was rugged and sensitive with a lower limit of quantification (LLOQ) of 0.5 ng/mL. All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. This method was successfully applied to evaluate the pharmacokinetics of d-amphetamine in rat. On a more general extent, this work demonstrated that the selectivity of the fragmentation pathway (MS³) can be used as alternative approach to significantly improve detection capability in complex situation (e.g., small molecules in complex matrices) rather than increasing time for sample preparation and chromatographic separation.     

Development,validation of a GC–MS method for the simultaneous measurement of amino acids,their PTM metabolites and AGEs in human urine,and application to the bi-ethnic ASOS study with special emphasis to lysine

A gas chromatography-mass spectrometry (GC–MS) method was developed and validated in relevant concentration ranges for the simultaneous measurement of l-lysine (Lys, L) and its N^ε- and N^α-methylated (M), N^ε- and N^α-acetylated (Ac), N^ε-carboxymethylated (CM) and N^ε-carboxyethylated (CE) metabolites in human urine. Analyzed Lys metabolites were the post-translational modification (PTM) products N^ε-mono-, di- and trimethyllsine, N^ε-MML, N^ε-DML, N^ε-TML, respectively, N^α-ML, N^ε-AcL, N^α-AcL, and its advanced glycation end-products (AGEs) N^ε-CML, N^ε-CM-[2,4,4-²H₃]Lys (d₃-CML), N^ε-CEL and furosine. AGEs of arginine (Arg) and cysteine (Cys) were also analyzed. De novo synthesized trideutero-methyl esters (R-COOCD₃) from unlabelled amino acids and derivatives were used as internal standards. Native urine samples (10 µL aliquots) were evaporated to dryness under a stream of nitrogen. Analytes were esterified using 2 M HCl in methanol (60 min, 80 °C) and subsequently amidated by pentafluoropropionic anhydride in ethyl acetate (30 min, 65 °C). The generated methyl ester-pentafluoropropionyl (Me-PFP) derivatives were reconstituted in borate buffer and extracted immediately with toluene. GC–MS analyses were performed by split-less injection of 1-µL aliquots, oven-programmed separation and negative-ion chemical ionization (NICI). Mass spectra were generated in the scan mode (range, m/z 50–1000). Quantification was performed in the selected-ion monitoring (SIM) mode using a dwell time of 50 or 100 ms for each ion. The GC–MS method was suitable for the measurement of Lys and all of its metabolites, except for the quaternary ammonium cation N^ε-TML. The Me-PFP derivatives of Lys, Arg and Cys and its metabolites eluted in the retention time window of 9 to 14 min. The derivatization of N^ε-CML, d₃-CML and N^ε-CEL was accompanied by partial N^ε-decarboxylation and formation of the Me-PFP Lys derivative. The lowest derivatization yield was observed for N^ε-DML, indicating a major role of the N^ε-DML group in Lys derivatization. The GC–MS method enables precise (relative standard deviation, RSD?<?20%) and accurate (bias,?<?±?20%) simultaneous measurement of 33 analytes in human urine in relevant concentration ranges. We used the method to measure the urinary excretion rates of Lys and its PTM metabolites and AGEs in healthy black (n?=?39) and white (n?=?41) boys of the Arterial Stiffness in Offspring Study (ASOS). No remarkable differences were found indicating no ethnic-related differences in PTM metabolites and AGEs except for N^ε-monomethyllysine and S-(2-carboxymethylcysteine).

     

Development and validation of a sensitive ultra performance liquid chromatography tandem mass spectrometry method for the analysis of fentanyl and its major metabolite norfentanyl in urine and whole blood in forensic context

Fentanyl and its major metabolite norfentanyl often occur in low doses in biological samples. Therefore, a highly sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed and fully validated. Sample preparation was performed on a mixed-mode cation exchange solid phase extraction (SPE) cartridge with an additional alkaline wash step to decrease matrix effects and thus increase sensitivity. Ionization of fentanyl and norfentanyl with electrospray ionization (ESI) was more efficient than atmospheric pressure chemical ionization (APCI). The use of a mobile phase of high pH resulted in higher ESI signals than the conventional low pH mobile phases. In the final method, gradient elution with 10 mM ammonium bicarbonate (pH 9) and methanol was performed. A comparison of columns with different internal diameter and/or smaller particles showed optimal resolution and sensitivity when an Acquity C18 column (1.7 μm, 2.1 mm × 50 mm) was used. Deuterium labeled internal standards were used, but with careful evaluation of their stability since loss of deuteriums was seen. With limits of detection of 0.25 pg/ml for fentanyl and 2.5 pg/ml for norfentanyl in urine and 5 pg/ml for fentanyl and norfentanyl in whole blood the presented method is highly appropriate for the analysis of fentanyl and norfentanyl in forensic urine and blood samples.