Use of Nonhuman Sera as a Highly Cost-Effective Internal Standard for Quantitation of Multiple Human Proteins Using Species-Specific Tryptic Peptides: Applicability in Clinical LC-MS Analyses.

Quantitation of proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is complex, with a multiplicity of options ranging from label-free techniques to chemically and metabolically labeling proteins. Increasingly, for clinically relevant analyses, stable isotope-labeled (SIL) internal standards (ISs) represent the "gold standard" for quantitation due to their similar physiochemical properties to the analyte, wide availability, and ability to multiplex to several peptides. However, the purchase of SIL-ISs is a resource-intensive step in terms of cost and time, particularly for screening putative biomarker panels of hundreds of proteins. We demonstrate an alternative strategy utilizing nonhuman sera as the IS for quantitation of multiple human proteins. We demonstrate the effectiveness of this strategy using two high abundance clinically relevant analytes, vitamin D binding protein [Gc globulin] (DBP) and albumin (ALB). We extend this to three putative risk markers for cardiovascular disease: plasma protease C1 inhibitor (SERPING1), annexin A1 (ANXA1), and protein kinase, DNA-activated catalytic subunit (PRKDC). The results show highly specific, reproducible, and linear measurement of the proteins of interest with comparable precision and accuracy to the gold standard SIL-IS technique. This approach may not be applicable to every protein, but for many proteins it can offer a cost-effective solution to LC-MS/MS protein quantitation.

Simultaneous quantification of cyclosporin, tacrolimus, sirolimus and everolimus in whole blood by UHPLC-MS/MS for therapeutic drug monitoring.

The aim of this study was to develop and validate a UHPLC-MS/MS assay to quantify cyclosporin (CYC), tacrolimus (TAC), sirolimus (SIR) and everolimus (EVE) in human whole blood for therapeutic drug monitoring. Analytes were extracted from 50 µL human whole blood by protein precipitation. The separation of the drugs was performed on an Acquity UPLC BEH C18 column. Analytes were eluted with a mobile phase consisting of 2 mM ammonium acetate with 0.1% formic acid (v/v) in deionised water and 2 mM ammonium acetate with 0.1% formic acid (v/v) in methanol at a flow rate of 300 µL/min in gradient elution. The method performance was evaluated by analysing patient blood samples and/or external quality control samples [proficiency testing (PT) scheme]. The method was linear from 23.75 to 1094.0, 1.3 to 42.4, 1.3 to 47.0 and 1.2-41.6 µg/mL for CYC, TAC, SIR and EVE, respectively. The within- and between-assay reproducibility results were ˂ 11%. Results from PT and patient sample quantification were comparable to those obtained previously by an in-house validated method using protein precipitation and liquid-liquid extraction. This method showed good analytical performance for quantifying CYC, TAC, SIR and EVE in whole blood over their respective calibration ranges.

Quantification of mycophenolic acid in human plasma by liquid chromatography with time-of-flight mass spectrometry for therapeutic drug monitoring.

This study presents, for the first time, the development and validation of a liquid chromatography and time-of-flight mass-spectrometry (LC-TOF-MS) based assay to quantify mycophenolic acid (MPA) in patient samples as part of a routine therapeutic drug monitoring service. MPA was extracted from 50 µl human plasma by protein precipitation, using sulindac as internal standard (IS). Separation was obtained on a Luna™ Omega polar C18 column kept at 40°C. The mobile phase consisted of a mixture of acetonitrile-deionized water (50:50, v/v) with 0.1% formic acid at a flow rate of 350 µl/min. Analyte and IS were monitored on a TOF-MS using a Jet-Stream™ (electrospray) interface running in positive mode. Assay performance was evaluated by analysing patient plasma (N = 69) and external quality assessment (N = 6) samples. The retention times were 2.66 and 2.18 min for MPA and IS, respectively. The lower limit of quantification of MPA was 0.1 µg/ml. The within- and between-assay reproducibility results ranged from 1.81 to 10.72%. Patient and external quality assessment sample results were comparable with those obtained previously by an in-house validated LC-MS/MS method. This method showed satisfactory analytical performance for the determination of MPA in plasma over the calibration range of 0.1-15.0 µg/ml.

Rapid Quantitation of Flecainide in Human Plasma for Therapeutic Drug Monitoring Using Liquid Chromatography and Time-of-Flight Mass Spectrometry.

BACKGROUND: Measurement of flecainide is useful to optimize dosage and minimize risks of toxicity. Furthermore, there is a need for urgent sample analysis when flecainide is used in transplacental therapy for fetal tachycardia. To this end, we have developed and validated a rapid assay for the measurement of flecainide in human plasma or serum, using a small sample volume (50 µL). METHODS: After a simple deproteination with zinc sulfate and methanol, prepared samples were injected onto a short (30 mm) analytical column and eluted using a rapid gradient elution. Detection was performed using time-of-flight mass spectrometry. Flecainide was quantified using flecainide-D4 as internal standard, with both compounds extracted from the total ion chromatogram using a ±5 ppm extraction window based on the theoretical m/z values for the protonated ions. RESULTS: The assay was linear over a putative therapeutic range (100-1500 mcg/L). Between- and within-assay imprecision and accuracy were <4.6% and 94.8%-110.0%, respectively. Matrix effects were minimal and were compensated for by flecainide-D4. There were no effects due to hemolysis or lipemia, and no carryover was apparent. Total analysis time was just 1.2 minutes (72 seconds). CONCLUSIONS: We have developed and validated a rapid method for the analysis of flecainide. The method is particularly suited for flecainide therapeutic drug monitoring, when analyzing samples from mothers receiving flecainide for the treatment of fetal tachycardia.

A 13-Steroid Serum Panel Based on LC-MS/MS: Use in Detection of Adrenocortical Carcinoma.

BACKGROUND: Adrenocortical carcinoma (ACC) is a rare malignancy, with an annual incidence of 1 or 2 cases per million. Biochemical diagnosis is challenging because up to two-thirds of the carcinomas are biochemically silent, resulting from de facto enzyme deficiencies in steroid hormone biosynthesis. Urine steroid profiling by GC-MS is an effective diagnostic test for ACC because of its capacity to detect and quantify the increased metabolites of steroid pathway synthetic intermediates. Corresponding serum assays for most steroid pathway intermediates are usually unavailable because of low demand or lack of immunoassay specificity. Serum steroid analysis by LC-MS/MS is increasingly replacing immunoassay, in particular for steroids most subject to cross-reaction. METHODS: We developed an LC-MS/MS method for the measurement of serum androstenedione, corticosterone, cortisol, cortisone, 11-deoxycorticosterone, 11-deoxycortisol, 21-deoxycortisol, dehydroepiandrosterone sulfate, pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, and testosterone. Assay value in discriminating ACC from other adrenal lesions (phaeochromocytoma/paraganglioma, cortisol-producing adenoma, and lesions demonstrating no hormonal excess) was then investigated. RESULTS: In ACC cases, between 4 and 7 steroids were increased (median = 6), and in the non-ACC groups, up to 2 steroids were increased. 11-Deoxycortisol was markedly increased in all cases of ACC. All steroids except testosterone in males and corticosterone and cortisone in both sexes were of use in discriminating ACC from non-ACC adrenal lesions. CONCLUSIONS: Serum steroid paneling by LC-MS/MS is useful for diagnosing ACC by combining the measurement of steroid hormones and their precursors in a single analysis.

Triazole antifungals used for prophylaxis and treatment of invasive fungal disease in adult haematology patients: Trough serum concentrations in relation to outcome.

Triazole antifungal drugs are widely used for the prophylaxis and treatment of invasive fungal disease (IFD). Efficacy may depend on attaining minimum effective plasma concentrations. The aim of this study was to ascertain the proportion of samples in which the recommended concentrations were achieved in patients given these drugs in relation to outcome. In-patients prescribed standard doses of fluconazole, itraconazole solution, posaconazole suspension, or oral voriconazole for at least one week were studied. Pre-dose serum triazole concentrations were measured using validated methods. There were 359 samples from 90 patients. The median (range) number of samples per patient was 3 (1-13), and the median (range) fluconazole, itraconazole, posaconazole (prophylaxis), posaconazole (treatment), and voriconazole serum concentrations were 5.64 (0.11-18), 0.57 (0-5.3), 0.31 (0.02-2.5), 0.65 (0.02-2.5), and 0.95 (0.10-5.4) mg/l, respectively. The number of samples in which the recommended pre-dose concentrations were achieved was 98 (54%), 9 (20%), 2 (18%), and 29 (49%) for itraconazole, posaconazole (>0.7 mg/l prophylaxis), posaconazole (treatment), and voriconazole, respectively. No significant differences were detected in the median triazole trough concentrations between patients with proven/probable IFD compared to those with no evidence of IFD. However, itraconazole was not detected in 10 samples (7 patients). The small number of patients who achieved the recommended trough posaconazole concentrations may explain the high rate of break-through IFD observed in patients prescribed this drug. Except for fluconazole, the number of patients prescribed standard doses of triazoles who achieved recommended trough triazole concentrations was low. The prospective use of serum triazole measurements assay may have improved outcomes with itraconazole, posaconazole, and with voriconazole.

Automated Analysis of Clozapine and Norclozapine in Human Plasma Using Novel Extraction Plate Technology and Flow-Injection Tandem Mass Spectrometry.

BACKGROUND: Analysis of plasma clozapine and N-desmethylclozapine (norclozapine) for therapeutic drug monitoring purposes is well established. To minimize analysis times and facilitate rapid reporting of results, we have fully automated sample preparation using novel AC Extraction Plates and a Tecan Freedom EVO 100 liquid handling platform, and minimized extract analysis times using flow-injection tandem mass spectrometry (FIA-MS/MS). METHODS: Analytes and deuterium-labeled internal standards were extracted from plasma (100 µL) at pH 10.6 and extracts analyzed directly using tandem mass spectrometry [20 µL injection, 0.7 mL/min methanol carrier flow, analysis time (injection-to-injection) approximately 60 seconds]. RESULTS: Validation data showed excellent intraplate and interplate accuracy (95%-104% nominal concentrations). Interbatch precision (% RSD) at the limit of quantitation (0.01 mg/L) was 3.5% and 5.5% for clozapine and norclozapine, respectively. Matrix effects were observed for both clozapine and norclozapine, but were compensated for by the internal standards. Overall process efficiency was 56%-70% and 66%-77% for clozapine and norclozapine, respectively. Mean relative process efficiency was 98% and 99% for clozapine and norclozapine, respectively. Comparison of results from patient samples (n = 81) analyzed using (1) manual liquid-liquid extraction with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and (2) automated extraction with FIA-MS/MS gave y = 1.01x - 0.002, R(2) = 0.9943 and y = 1.01x + 0.009, R(2) = 0.9957 for clozapine and norclozapine, respectively. Bland-Altman plots revealed a [mean (95% limits of agreement) bias of 0.0074 (-0.04 to 0.06) mg/L and of 0.015 (-0.02 to 0.05) mg/L for clozapine and norclozapine, respectively]. CONCLUSIONS: FIA-MS/MS used with automated extraction offers a rapid, simple, cost-effective alternative to manual liquid-liquid extraction and conventional LC analysis for clozapine therapeutic drug monitoring.

Sex differences in plasma clozapine and norclozapine concentrations in clinical practice and in relation to body mass index and plasma glucose concentrations: a retrospective survey.

BACKGROUND: Clozapine is widely prescribed and, although effective, can cause weight gain and dysglycemia. The dysmetabolic effects of clozapine are thought to be more prevalent in women with this gender on average attaining 17 % higher plasma clozapine concentrations than men. METHODS: We investigated the relationship between dose, body mass index (BMI), plasma glucose concentration, and plasma clozapine and N-desmethylclozapine (norclozapine) concentrations in 100 individuals with a severe enduring mental illness. RESULTS: Mean (10th/90th percentile) plasma clozapine concentrations were higher for women [0.49 (0.27-0.79) mg/L] compared with men [0.44 (0.26-0.70) mg/L] (F = 2.2; p = 0.035). There was no significant gender difference in the prescribed clozapine dose. BMI was significantly higher in women [mean (95 % CI) = 34.5 (26.0-45.3)] for females compared with 32.5 (25.2-41.0) for males. Overall, BMI increased by 0.7 kg/m(2) over a mean follow-up period of 210 days. A lower proportion, 41 % of women had a fasting blood glucose ≤6.0 mmol/L (<6.0 mmol/L is defined by the International Diabetes Federation as normal glucose handling), compared with 88 % of men (χ (2) = 18.6, p < 0.0001). CONCLUSIONS: We have shown that mean BMI and blood glucose concentrations are higher in women prescribed clozapine than in men. Women also tended to attain higher plasma clozapine concentrations than men. The higher BMI and blood glucose in women may relate to higher tissue exposure to clozapine, as a consequence of sex differences in drug metabolism.

An automated, high-throughput method for targeted quantification of intact insulin and its therapeutic analogs in human serum or plasma coupling mass spectrometric immunoassay with high resolution and accurate mass detection (MSIA-HR/AM).

The detection and quantification of insulin and its therapeutic analogs is important for medical, sports doping, and forensic applications. Synthetic variants contain slight sequence variations to affect bioavailability. To reduce sample handling bias, a universal extraction method is required for simultaneous extraction of endogenous and variant insulins with subsequent targeted quantification by LC-MS. A mass spectrometric immunoassay (MSIA), a multiplexed assay for intact insulin and its analogues that couples immunoenrichment with high resolution and accurate mass (HR/AM) spectrometric detection across the clinical range is presented in this report. The assay is sensitive, selective, semi-automated and can potentially be applied to detect new insulin isoforms allowing their further incorporation into second or third generation assays.

Measurement of the direct oral anticoagulants apixaban, dabigatran, edoxaban, and rivaroxaban in human plasma using turbulent flow liquid chromatography with high-resolution mass spectrometry.

BACKGROUND: Direct oral anticoagulants (DOACs) are prescribed for systemic anticoagulation. Fixed doses are recommended, but dose individualization may be warranted. Functional coagulation assays may be available, but their use requires knowledge of the drug taken. To provide alternative methodology for guiding dosage, we have developed and validated a liquid chromatography-mass spectrometric assay for apixaban, dabigatran, edoxaban, and rivaroxaban at the concentrations attained during therapy. METHODS: Samples, calibrators, and internal quality controls (100 µL) were mixed with internal standard solution (50 µg/L both dabigatran-13C6 and rivaroxaban-13C6 in acetonitrile) and, after centrifugation (16,400g, 4 minutes), supernatant (100 µL) was injected onto a Cyclone-C18-P-XL TurboFlow column. Analytes were focused onto an Accucore PhenylHexyl (2.1 × 100 mm, 2.6 µm) analytical column and eluted using a methanol + acetonitrile (1 + 1):aqueous ammonium acetate (10 mmol/L) gradient. Data were acquired using high-resolution mass spectrometry in full-scan mode (100-2000 m/z) with data-dependent fragmentation to confirm peak identity. Calibration was linear (1-500 µg/L all analytes). RESULTS: Total analysis time was 6 minutes. Intra-assay imprecision (% RSD) at 1 µg/L was 2.6%, 4.2%, 17.3%, and 9.5% for apixaban, dabigatran, edoxaban, and rivaroxaban, respectively. Mean recovery was 96%-101%. No signal suppression or enhancement was observed. Apixaban, dabigatran, and rivaroxaban were stable over 3 freeze-thaw cycles, after storage at room temperature, and at 2-8°C for up to 2 weeks. Edoxaban was stable over 3 freeze-thaw cycles but showed a mean deterioration of 16% if stored at 2-8°C (2 weeks) and of 18% and 70% (1 day and 2 weeks, respectively) at room temperature. CONCLUSIONS: The method is suitable for high-throughput therapeutic drug monitoring of DOACs. The acquisition of full scan data allows for the retrospective identification of metabolites. The method can be used to identify a particular DOAC if information on the drug taken is lacking.

LC-MS candidate reference methods for the harmonisation of parathyroid hormone (PTH) measurement: a review of recent developments and future considerations.

The analysis of intact parathyroid hormone (PTH) (PTH1-84) is useful in the diagnosis of hyper- and hypocalcaemia, hyperparathyroidism, and in the prevention of bone mineral disorders in renal patients. The analysis is complicated by the presence of PTH fragments, which may accumulate in renal failure and cross-react in immunoassays, including the most recent third-generation immunoassays. Large variability exists between different commercially available assays. This article reviews the current literature on PTH testing, with emphasis on the use of mass spectrometry-based methods, and considers the important sources of variation which still need to be addressed prior to the development of much needed candidate reference methods for PTH analysis. Recently, mass spectrometric methods have been developed for the quantitation of PTH1-84 using surrogate tryptic peptides, but even these methods are subject to significant interferences due to the presence of newly observed modified PTH species, such as oxidised and phosphorylated PTH variants, which can accumulate in patient samples. Further work, including: 1) the use of high-resolution mass spectrometry; and 2) the analysis of PTH without prior protease digestion, is required before these approaches can be considered as reference methods against which other methods should be harmonised.

Analysis of drug-impregnated paper samples seized in English prisons between 2018 and 2020.

Novel psychoactive substances (NPS) in the form of impregnated papers delivered to prisoners are of particular concern in prison settings, where they are commonly used by vaping. The purpose of this study was to create a qualitative method for identifying the various emerging NPS impregnated onto paper samples sent to prisoners. It helps to demonstrate that these findings can be used to predict drug prevalence and trends in prisons. Between 2018 and 2020, 1250 non-judicial paper samples seized from 12 English prisons were analysed to determine the NPSs being circulated. Approximately 1 cm2 paper were cut and added to 50 % (v/v) methanol in LCMS-grade water. Vortex-mixing was used to prepare extracts (30 min). Q-TOF LC/MS was used to screen the extracts. This study showed that synthetic cannabinoid receptor agonist (SCRA) was the most common drug group detected in impregnated paper seizures in English prisons between 2018 and 2020, followed by cocaine, heroin type drugs (A) and amphetamine, ketamine type drugs (B). Male prisons had a higher prevalence of SCRAs, whereas female prisons had a higher prevalence of A drugs. Furthermore, lower security prisons were found to have a higher prevalence of B drugs, pregabalin, gabapentin type drugs (C), and abused and prescription drugs than higher security prisons which unveiled a higher prevalence of nicotine. The findings of this study have revealed new information about drug use in prisons. This study will also aid in the identification of drug smuggling routes into jails, keeping prison staff up to date with the trends.

Analysis of drugs seized from amnesty bins at two major United Kingdom summer music festivals using two portable gas chromatography-mass spectrometry (GC-MS) instruments.

Globally, the number of drug users and the proportion of the drug using population has increased from 210 million in 2009 to 269 million in 2019. Several studies suggest that music festival attendees are more likely to abuse illicit substances and have a high-risk profile. Consequently, it is crucial to develop robust field drug analysis methods that facilitate harm reduction and drug monitoring. The work presented in this report aimed at developing and validating qualitative analytical methods for 3,4-methylenedioxymethamphetamine, 4-bromo-2,5-dimethoxyphenethylamine (2C-B), ketamine and N-ethylpentylone on two portable gas chromatography-mass spectrometry (GC-MS) systems: Griffin G510 (Teledyne FLIR, West Lafayette, IN) and Torion T-9 (PerkinElmer, Shelton, CT). The diagnostic ability of the mobile GC-MS units was assessed on 200 samples in total, seized at two large summer music festivals in the United Kingdom. The method validation process included selectivity/specificity, limit of identification, carry-over, ruggedness/robustness, and inter- and intra-day precision (repeatability and reproducibility). The Griffin G510 demonstrated a limit of identification from 1 mg/mL for 2C-B to 0.063 mg/mL for ketamine and good ruggedness and precision results. The precision for 2C-B using the Torion T-9 was poorer than for the Griffin G510, but equivalent for the other compounds tested. Correct identifications (versus benchtop GC-MS) for the two festivals were 85%-86% and 74%-83% for the Griffin G510 and the Torion T-9, respectively. The two portable instruments were able to adequately cover current on-site drug-testing analytical gaps and proved to be a powerful addition to the on-site drug analysis techniques.

Assessment of a Single Quadrupole Mass Spectrometer Combined with an Atmospheric Solids Analysis Probe for the On-Site Identification of Amnesty Bin Drugs.

The surging number of people who abuse drugs has a great impact on healthcare and law enforcement systems. Amnesty bin drug analysis helps monitor the "street drug market" and tailor the harm reduction advice. Therefore, rapid and accurate drug analysis methods are crucial for on-site work. An analytical method for the rapid identification of five commonly detected drugs ((3,4-methylenedioxymethamphetamine (MDMA), cocaine, ketamine, 4-bromo-2,5-dimethoxyphenethylamine, and chloromethcathinone)) at various summer festivals in the U.K. was developed and validated employing a single quadrupole mass spectrometer combined with an atmospheric pressure solids analysis probe (ASAP-MS). The results were confirmed on a benchtop gas chromatography-mass spectrometry instrument and included all samples that challenged the conventional spectroscopic techniques routinely employed on-site. Although the selectivity/specificity step of the validation assessment of the MS system proved a challenge, it still produced 93% (N = 279) and 92.5% (N = 87) correct results when tested on- and off-site, respectively. A few "partly correct" results showed some discrepancies between the results, with the MS-only unit missing some low intensity active ingredients (N-ethylpentylone, MDMA) and cutting agents (caffeine, paracetamol, and benzocaine) or detecting some when not present. The incorrect results were mainly based on library coverage. The study proved that the ASAP-MS instrument can successfully complement the spectroscopic techniques used for qualitative drug analysis on- and off-site. Although the validation testing highlighted some areas for improvement concerning selectivity/specificity for structurally similar compounds, this method has the potential to be used in trend monitoring and harm reduction.

Broad evidence of xylazine in the UK illicit drug market beyond heroin supplies: Triangulating from toxicology, drug-testing and law enforcement.

BACKGROUND AND AIMS: Xylazine is a non-opioid sedative which has spread rapidly throughout the US illicit drug supply. This study aimed to describe the spread of xylazine throughout the UK illicit drug supply. METHODS: Xylazine detections in human biological samples were collated from toxicology laboratories operating in the United Kingdom with the date, location, case type, xylazine concentration and co-detected drugs (with quantifications where performed) detailed, where permitted, by the corresponding coroner. Drug-testing cases positive for xylazine were collated from the Welsh Emerging Drugs and Identification of Novel Substances (WEDINOS) drug-testing postal service with the date, location, purchase intent and co-detected drugs detailed. Drug seizures made by UK law enforcement were communicated by the Office for Health Improvement and Disparities with the date and location detailed. RESULTS: By the end of August 2023, xylazine was detected in 35 cases from throughout toxicology, drug-testing and drug seizure sources covering England, Scotland and Wales. There were no cases reported from Northern Ireland. Xylazine was detected in biological samples from 16 people. In most cases where full toxicology results were provided, xylazine was detected with heroin and/or a strong opioid (n = nine of 11), but this polydrug use pattern was not evident in all cases (n = two of 11), suggesting a wider circulation of xylazine in the UK illicit drug market beyond heroin supplies. Evidence from WEDINOS supports this claim, as all 14 drug samples (100%) submitted from across the UK contained xylazine; however, in none of these cases was heroin the purchase intent but rather counterfeit prescription medication tablets (n = 11 of 14), tetrahydrocannabinol (THC) vapes (n = two of 14) or white powder (n = one of 14). Additional evidence for the spread of illicit xylazine comes from five drug seizures made by law enforcement. CONCLUSIONS: Xylazine has penetrated the UK illicit drug market and is not limited to heroin supplies.

A novel approach to quantitative LC-MS/MS: therapeutic drug monitoring of clozapine and norclozapine using isotopic internal calibration.

Therapeutic drug monitoring (TDM) requires timely results in order to be clinically helpful. Such assays, when carried out using mass spectrometry-based methods, typically involve a batched sample approach with multipoint calibration. Isotopic internal calibration offers the possibility of open-access mass spectrometric analysis with consequent shortening of turnaround times. We measured plasma clozapine and N-desmethylclozapine (norclozapine) concentrations in (1) external quality assessment (EQA) samples (N = 22) and (2) patient samples (N = 100) using liquid chromatography-tandem mass spectrometry with isotopic internal calibration (ICAL-LC-MS/MS). Analyte concentrations were calculated from graphs of the response of three internal calibrators (clozapine-D4, norclozapine-D8, and clozapine-D8) against concentration. Precision (% RSD) and accuracy (% nominal concentrations) for the ICAL-LC-MS/MS method were <5 % and 104-112 %, respectively for both analytes. There was excellent agreement with consensus mean and with 'spiked' values on analysis of the EQA samples (R (2) = 0.98 and 0.97, respectively, inclusive of clozapine and norclozapine results). In the patient samples, comparison against traditionally calibrated HPLC-UV and LC-MS/MS methods showed excellent agreement (R (2) = 0.97 or better) with small albeit significant mean differences (<0.041 and <0.042 mg/L for clozapine and norclozapine, respectively). These differences probably reflect discrepancies in the in-house preparation of calibrators and/or interference in the UV method. Internal calibration offers a novel and attractive alternative to traditionally calibrated batch analysis in analytical toxicology. The method described has been validated for use in the high-throughput TDM of clozapine and norclozapine, and allows for (1) same-day reporting of results and (2) significant cost savings.