Quantification and application of a liquid chromatography–tandem mass spectrometric method for the determination of WKYMVm peptide in rat using solid‐phase extraction

A liquid chromatographic–electrospray ionization–time‐of‐flight/mass spectrometric (LC‐ESI‐TOF/MS) method was developed and applied for the determination of WKYMVm peptide in rat plasma to support preclinical pharmacokinetics studies. The method consisted of micro‐elution solid‐phase extraction (SPE) for sample preparation and LC‐ESI‐TOF/MS in the positive ion mode for analysis. Phenanthroline (10 mg/mL) was added to rat blood immediately for plasma preparation followed by addition of trace amount of 2 m hydrogen chloride to plasma before SPE for stability of WKYMVm peptide. Then sample preparation using micro‐elution SPE was performed with verapamil as an internal standard. A quadratic regression (weighted 1/concentration²), with the equation y = ax² + bx + c was used to fit calibration curves over the concentration range of 3.02–2200 ng/mL for WKYMVm peptide. The quantification run met the acceptance criteria of ±25% accuracy and precision values. For quality control samples at 15, 165 and 1820 ng/mL from the quantification experiment, the within‐run and the between‐run accuracy ranged from 92.5 to 123.4% with precision values ≤15.1% for WKYMVm peptide from the nominal values. This novel LC‐ESI‐TOF/MS method was successfully applied to evaluate the pharmacokinetics of WKYMVm peptide in rat plasma.     

Evaluation of dried blood spot (DBS) technology versus plasma analysis for the determination of MK-1775 by HILIC-MS/MS in support of clinical studies

The collection of human blood samples as dried blood spots (DBS) for the pharmacokinetic assessment of investigational drugs in clinical trials offers a number of advantages over conventional plasma sampling, namely, small sample volume, simplified sample handling, and cost-effective shipping and storage. The use of DBS coupled with liquid chromatography–tandem mass spectrometry analysis was evaluated for the quantification of MK-1775, a Wee-1 inhibitor under development as a chemo/radio-sensitizer for the treatment of cancer. The DBS method exhibited an assay performance comparable to that of the existing plasma assay, which is currently used in support of clinical studies. Both assays used the same linear dynamic range of 2–1,000?ng/mL, with a lower limit of quantification of 2?ng/mL. Based on the intra-day assay validation results, the accuracy of the DBS method ranged from 94.0 to 105.0?%, with a coefficient of variation of <4.8?%. The blood-to-plasma ratio calculated from the DBS data (blood concentrations) and the plasma data (plasma concentrations) was in good agreement with the one obtained from the in vitro assessment using conventional methodology. No significant hematocrit impact on the assay was observed as hematocrit ranged from 16 to 85?%. The correlation between the measured MK-1775 concentrations in plasma and that determined in dried blood spots from oncology patients during the ongoing clinical study was discussed.     

Development and application of a new on-line SPE system combined with LC-MS/MS detection for high throughput direct analysis of pharmaceutical compounds in plasma

A technique using a fully automated on-line solid phase extraction (SPE) system (Symbiosis, Spark Holland) combined with liquid chromatography (LC)-mass spectrometry (MS/MS) has been investigated for fast bioanalytical method development, method validation and sample analysis using both conventional C18 and monolithic columns. Online SPE LC-MS/MS methods were developed in the automated mode for the quantification of model compounds (propranolol and diclofenac) directly in rat plasma. Accuracy and precision using online SPE LC-MS/MS with conventional C18 and monolithic columns were in the range of 88-111% and 0.5-14%, respectively. Total analysis cycle time of 4 min per sample was demonstrated using the C18 column. Monolithic column allowed for 2 min total cycle time without compromising the quality and validation criteria of the method. Direct plasma sample injection without on-line SPE resulted in poor accuracy and precision in the range of 41-108% and 3-81%. Furthermore, the increase in back pressure resulted in column damage after the injection of only 60 samples.     

Liquid chromatography–electrospray ionization tandem mass spectrometry for the quantification of styraxlignolide A in rat plasma

Styraxlignolide A is a pharmacologically active ingredient isolated from Styrax japonica Sieb. et Zucc. A rapid, selective, and sensitive liquid chromatographic method with electrospray ionization tandem mass spectrometry was developed for use in the quantification of styraxlignolide A in rat plasma. Styraxlignolide A was extracted from rat plasma using ethyl acetate at neutral pH. The analytes were separated on an Atlantis dC₁₈ column using a mixture of methanol and ammonium formate (10 mM, pH 3.0) (70:30, v/v) and detected by tandem mass spectrometry in multiple reaction monitoring mode. The standard curve was linear (r²=0.9978) over the concentration range of 100?10000 ng/mL. The lower limit of quantification was 100 ng/mL using 50 μL of plasma sample. The coefficient of variation and relative error for intra‐ and inter‐assays at four QC levels were 1.6–8.3% and from ?12.0 to ?1.7%, respectively. The present method was applied successfully to the pharmacokinetic study of styraxlignolide A after intravenous administration of styraxlignolide A at a dose of 10 mg/kg in male Sprague–Dawley rats.     

Development of a stable isotope dilution UPLC‐MS/MS method for quantification of dexmedetomidine in a small amount of human plasma

Dexmedetomidine (Dex) is a selective central α2‐agonist with anesthetic properties and has been used in clinical practice for sedation in the intensive care unit (ICU) after operations. In this study, an analytical assay for the determination of Dex in a small amount of plasma was developed for the application to pediatric ICU trials. The quantification of Dex was constructed using the original stable isotope Dex‐d₃ for electrospray ionization‐tandem mass spectrometry (ESI‐MS/MS) in the selected reaction monitoring mode. A rapid ultra‐performance liquid chromatography technique was adopted using ESI‐MS/MS with a runtime of 3 min. Efficacious concentration levels (50 pg/mL to 5 ng/mL) could be evaluated using a very small amount of plasma (10 μL) from patients. The lower limit of the quantification was 5 pg/mL in the plasma (100 µL). For sample preparation, a solid‐phase extraction was used along with the OASIS‐HLB cartridge type. Recovery values ranged from 98.8 to 100.3% for the intra‐ [relative standard deviation (RSD), 0.9–1.3%] and inter‐ (RSD, 0.9–1.5%) day assays. A stable test had recovery values that ranged from 97.8 to 99.7% with an RSD of 1.0–1.9% for the process/wet extract, bench‐top, freeze–thaw and long‐term tests. This method was used to measure the Dex levels in plasma from pediatric ICU patients. In the clinical ICU trial, the small amount of blood (approximate plasma volume, 200 μL) remaining from blood gas analysis was reused and targeted for the clinical analysis of Dex in plasma. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous quantification of methylene blue and its major metabolite,azure B,in plasma by LC‐MS/MS and its application for a pharmacokinetic study

A simple and sensitive liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method was developed for the quantification of methylene blue (MB) and its major metabolite, azure B (AZB), in rat plasma. A simple protein precipitation using acetonitrile was followed by injection of the supernatant on to a Zorbax HILIC Plus column (3.5 µm, 2.1 × 100 mm) with isocratic mobile phase consisting of 5 mM ammonium acetate in 10:90 (v/v) water:methanol at a flow rate of 0.3 mL/min and detection in positive ionization mode. The standard curve was linear over the concentration range from 1 to 1000 ng/mL for MB and AZB with coefficient of determination above 0.9930. The lower limit of quantification was 1 ng/mL using 20 μL of rat plasma sample. The intra‐ and inter‐assay precision and accuracy were <12%. The developed analytical method was successfully applied to the pharmacokinetic study of MB and AZB in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of ramoplanin in human urine by high-performance liquid chromatography with automated column switching.

Ramoplanin is a novel glycolipodepsipeptide antibiotic, currently undergoing clinical trials. This method describes the determination of ramoplanin by direct injection of human urine into a coupled-column liquid chromatographic system. An internal-surface reversed-phase column has been used for on-line sample clean-up and enrichment. Analytical separation of ramoplanin and MDL 62,456 used as internal standard, has been achieved on a ABZ+ reversed-phase column with ammonium acetate buffer-acetonitrile-methanol according to a gradient profile. Analytes were detected by their UV absorbance at 270 nm. The limit of quantitation was 0.1 microgram/ml urine and the limit of detection was found to be 0.035 microgram/ml, corresponding to 13.7 pmol/ml. Linearity was determined in the range 0.1-2 micrograms/ml. Precision (relative standard deviation) ranged from 0.71 to 8.75% and the accuracy from -9.9 to 11.6%. Different human sources were tested and no interference between analytes and urine constituents was observed. The method is simple and rapid, requiring a total analysis time of 35 min per sample and reaching greater selectivity and accuracy than microbiological assays.     

Simultaneous and rapid determination of gefitinib,erlotinib and afatinib plasma levels using liquid chromatography/tandem mass spectrometry in patients with non‐small‐cell lung cancer

A simultaneous, selective, sensitive and rapid liquid chromatography/tandem mass spectrometry method was developed and validated for the quantification of gefitinib, erlotinib and afatinib in 250 μL samples of human blood plasma. Diluted plasma samples were extracted using a liquid‐phase extraction procedure with tert‐butyl methyl ether. The three drugs were separated by high‐performance liquid chromatography using a C₁₈ column and an isocratic mobile phase running at a flow rate of 0.2 mL/min for 5 min. The drugs were detected using a tandem mass spectrometer with electrospray ionization using imatinib as an internal standard. Calibration curves were generated over the linear concentration range of 0.05–100 nm in plasma with a lower limit of quantification of 0.01 or 0.05 nm for all compounds. Finally, the validated method was applied to a clinical pharmacokinetic study in patients with nonsmall‐cell lung cancer (NSCLC) following the oral administration of afatinib. These results indicate that this method is suitable for assessing the risks and benefits of chemotherapy in patients with NSCLC and is useful for therapeutic drug monitoring for NSCLC treatment. As far as we know, this is the first report on LC‐MS/MS method for the simultaneous quantification of NSCLC tyrosine kinase inhibitor plasma concentrations including afatinib. Copyright © 2015 John Wiley & Sons, Ltd.     

Fragmentation of peptides with intra-chain disulfide bonds in triple quadrupole mass spectrometry and its quantitative application to biological samples

A growing number of peptides are being used today in bioanalytical laboratories. Because of this, there is an increasing interest in the development of highly sensitive, specific and robust liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays for the quantitative analysis of peptides in biological samples. Among the mass spectrometers previously used for peptide quantification, triple quadrupole mass spectrometers are generally not considered the instrument of choice. With this instrumentation, collision cascades or multiple fragmentations tend to generate multiple peaks that have weak intensities. This leads to a loss in detection sensitivity. However, in cases where immonium product ions were formed in abundance, it was found that peptide quantification succeeded. A common feature of these peptides is their intra-loop structure. To elucidate the usefulness of this feature in fragmentation, several peptide analytes with intra-chain disulfide bonds were investigated in this study, including a newly synthesized analog having a single amino acid substitution. The results presented here indicate that abrupt bond cleavage from the intra-loop structure of peptides could be one of the premises for intense immonium ion generation. In contrast, any preferential cleavage of peptide bonds (e.g., proline effect) that gives rise to a linearized sequence would break the intactness of the loop and prevent it from completely dissociating. In addition, the utilization of immonium product ions in LC/MS/MS was demonstrated for the determination of peptides with intra-chain disulfide bonds in biological fluids.     

LC-ESI-MS/MS determination of paclitaxel on dried blood spots

A simple and rapid high‐performance liquid chromatography–tandem mass spectrometric assay for determination of paclitaxel on rat dried blood spots was developed and validated. The extracted sample was chromatographed without further treatment using a reverse‐phase Oyster ODS3, 4.6 × 50 mm, 3 µm column with mass spectrometry detection. The mobile phase comprised of acetonitrile–water, 60:40 v/v, with a flow rate of 0.4 mL/min was used. The calibration was linear over the range 0.2–20 ng/mL. The limits of detection and quantification were 0.08 and 0.2 ng/mL, respectively. The intra‐ and inter‐day precision (CV%) and accuracy (relative error %) were less than 10 and 12%, respectively. Copyright © 2011 John Wiley & Sons, Ltd.     

Examples of doping control analysis by liquid chromatography-tandem mass spectrometry: ephedrines, beta-receptor blocking agents, diuretics, sympathomimetics, and cross-linked hemoglobins

The application of modern and powerful analytical instruments consisting of liquid chromatographs (LCs), sophisticated atmospheric pressure ion sources, and sensitive mass analyzers has improved quality as well as speed of doping control analyses markedly during the last 5 years. Numerous compounds such as beta-receptor blocking agents or diuretics require derivatization prior to gas chromatographic (GC) and mass spectrometric (MS) measurement, which is the reason for extended sample preparation periods. In addition, several substances demonstrate poor GC-MS properties even after chemical modification, and peptide hormones such as cross-linked hemoglobins cannot be analyzed at all by means of GC-MS. With the availability of electrospray ionization and robust tandem MSs (e.g., triple-stage quadrupole or ion trap instruments) many new or complementary screening and confirmation assays have been developed, providing detailed qualitative and quantitative information on prohibited drugs. With selected categories of compounds (ephedrines, beta-blockers, b2-agonists, diuretics, and bovine hemoglobin-based oxygen therapeutics) that are banned according to the rules of the World Anti-Doping Agency and International Olympic Committee, the advantages of LC-MS-MS procedures over conventional GC-MS assays are demonstrated, such as enhanced separation of analytes, shorter sample pretreatment, and identification of substances that are not identified by GC-MS.     

A rapid and sensitive liquid chromatography–tandem mass spectrometric assay for moexipril,an angiotensin‐converting enzyme inhibitor in human plasma

A simple, rapid and sensitive liquid chromatography/tandem mass spectrometry method was developed and validated for the quantification of angiotensin‐converting enzyme inhibitor, moexipril, in human plasma. Benazepril was used as an internal standard (IS). Analyte and IS were extracted from the human plasma by liquid–liquid extraction technique using ethyl acetate. The reconstituted samples were chromatographed on a C₁₈ column by using a mixture of methanol and 0.1% formic acid buffer (85:15, v/v) as the mobile phase at a flow rate of 0.5 mL/min. The calibration curve obtained was linear (r ≥ 0.99) over the concentration range of 0.2–204 ng/mL. The multiple reaction‐monitoring mode was used for quantification of ion transitions at m/z 499.4/234.2 and 425.2/351.1 for moexipril and IS, respectively. The results of the intra‐ and inter‐day precision and accuracy studies were well within the acceptable limits. A run time of 2.0 min for each sample made it possible to analyze more than 400 plasma samples per day. The proposed method was found to be applicable to clinical studies. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of telmisartan in human blood plasma: Part II: Liquid chromatography-tandem mass spectrometry method development, comparison to immunoassay and pharmacokinetic study

A new liquid chromatography/atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS) method with on-line sample clean-up for the determination of telmisartan in human blood plasma is presented. This technique is compared to a previously introduced enzyme-linked immunosorbent assay (ELISA), where fluorescence is used as detection method. For the LC/MS method applying an internal calibration via a deuterated internal standard, the limit of detection was 0.3 ng/mL, the limit of quantification was 0.9 ng/mL and the linear range extended from 0.9 to 1000 ng/mL. Forty-eight plasma samples from four healthy volunteers were analyzed in a pharmacokinetic study to obtain data for the method comparison. As a result, these two new and independent analytical methods for the determination of telmisartan in human blood plasma proved to yield comparable results for the amount of analyte.     

Development and validation of a liquid chromatography/tandem mass spectrometry assay for the quantification of methyl protodioscin in rat plasma: application to a pharmacokinetic study

A high performance liquid chromatography/tandem mass spectrometry assay was first developed and validated for the quantification of methyl protodioscin (MPD), a natural furostanol saponin with distinct antitumor activity, in rat plasma with 17alpha-ethinylestradiol as internal standard (IS). Methanol-mediated protein precipitation was employed for plasma sample pretreatment. The separation was achieved on a C(18) column (150 x 4.6 mm, i.d., 5 microm) by isocratic elution with methanol-water (72:28, v/v) as mobile phase at a flow rate of 1.0 mL/min. Ion acquisition was performed in selective reaction monitoring positive mode by monitoring the transition of m/z 1085.7 --> 1053.7 for MPD, and in selective ion monitoring negative mode by monitoring the deprotonated ion m/z 295.5 for IS. The assay was linear over the concentration range of 2.024-270.0 microg/mL with 2.024 microg/mL as the lower limit of quantification. It was specific, accurate, precise and reproducible with intra- and inter-run RSD <8.3% and RE between -11.5 and 12.8%. The assay was successfully applied to a preclinical pharmacokinetic study after an intravenous dose of 40 mg/kg MPD to rats.     

Ultrasensitive method to quantify intracellular zidovudine mono‐, di‐ and triphosphate concentrations in peripheral blood mononuclear cells by liquid chromatography–tandem mass spectrometry

Although zidovudine (AZT) is not the preferred antiretroviral drug for adult HIV‐infected patients, it is still widely used in infants for both prevention of mother‐to‐infant HIV‐1 transmission and treatment of HIV‐infected children. However, it is difficult to measure intracellular concentrations of AZT metabolites in small blood samples due to their extremely low concentrations in peripheral blood mononuclear cells and interference by endogenous nucleotide triphosphates, residual plasma phosphates and electrolytes. We developed an ultrasensitive assay using liquid chromatography–tandem mass spectrometry (LC–MS/MS) for measurement of intracellular concentrations of zidovudine (AZT)‐monophosphate (AZT‐MP), ‐diphosphate (AZT‐DP) and ‐triphosphate (AZT‐TP). The high sensitivity was due to the improvement of peripheral blood mononuclear cells extraction for complete removal of plasma and electrolytes, alkalization of LC buffer and use of alkaline‐stable high performance liquid chromatography column and tetrabutylammonium hydroxide as the ion pair. Using this method, the lower limits of quantification of AZT, AZT‐MP, ‐DP and ‐TP were 6, 6, 10 and 10 fmol per sample, respectively. Accuracy ranged 89–115% and precision was lower than 15% in the quantification range of 6–6000 fmol/sample for plasma AZT and intracellular AZT‐MP and 10–10 000 fmol/sample for AZT‐DP and ‐TP. The validation parameters met the international requirements. Among nine AZT‐treated HIV‐infected adult patients, five had low AZT‐TP levels (<10 fmol/10⁶ cells). Our assay has high sensitivity and is advantageous for evaluation of AZT phosphates in children and infants based on minimum blood sampling requirement. Copyright © 2015 John Wiley & Sons, Ltd.     

Unmasking low-abundance peptides from human blood plasma and serum samples by a simple and robust two-step precipitation/immunoaffinity enrichment method

Proteomic analysis of human plasma and serum for identifying and validating disease-specific marker proteins and peptides has one major drawback besides its unique advantage as a readily available sample source for diagnostic assays. This disadvantage is represented by the predominance of several high- and middle-abundant proteins, which clearly hamper identification and quantification approaches of potential and validated protein and peptide biomarkers, which are often of very low abundance. During the last decades, a significant number of depletion and enrichment techniques evolved to address these two issues. We present here a cost-effective and easy-to-use strategy for protein depletion comprising a thermal precipitation protocol followed by a two-step liquid/liquid precipitation as well as using an immunoaffinity chromatography method for the specific enrichment and isolation of the low-abundance polypeptide N-terminal pro-B-type natriuretic peptide and its precursor proBNP clinically used as biomarkers for the detection of severe human heart failure and related diseases. The applicability of this approach is shown by SDS -CGE, SDS-PAGE, electrochemiluminescence immunoassay and nano-LC ESI-MS/MS. Our thermal precipitation protocol followed by a two-step liquid/liquid precipitation could also serve as a potential depletion technique for the characterization of other low-abundance peptides and proteins.     

Development of a precise quantitative method for monitoring sirolimus in whole blood using LC/ESI–MS/MS

Sirolimus is used on patients after solid organ transplantation and on lymphangioleiomyomatosis (LAM) patients, and therapeutic drug monitoring is required in clinical practice. We have previously reported an accurate method for quantitative determination of sirolimus, but its sample preparation step was complicated. In this study, we developed a modified liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI–MS/MS) method for sirolimus quantification. A supported liquid extraction cartridge was used to purify sirolimus from whole blood and ion suppression was mostly prevented. The validation results met the acceptance criteria. This method was compared with the antigen conjugated magnetic immunoassay (ACMIA) and our previously reported method, using whole blood samples from LAM patients. Comparison of the Bland–Altman plots of the currently developed method and the previous method revealed no significant difference between the two methods (mean bias, −2.02%; 95% CI, −7.81–3.78). The values obtained using ACMIA were significantly higher than those obtained using the current method by 13.87% (95% CI, 6.49–21.25) owing to cross-reactivity. The degrees of cross reactivities in LAM patients and in organ transplant patients were similar, and our LC/ESI–MS/MS method precisely measured the blood concentrations of sirolimus.     

Novel rapid liquid chromatography tandem masspectrometry method for vemurafenib and metabolites in human plasma,including metabolite concentrations at steady state

A novel, rapid and sensitive liquid chromatography tandem–mass spectrometry method for quantification of vemurafenib in human plasma, that also for the first time allows for metabolite semi‐quantification, was developed and validated to support clinical trials and therapeutic drug monitoring. Vemurafenib was analysed by precipitation with methanol followed by a 1.9 min isocratic liquid chromatography tandem masspectrometry analysis using an Acquity BEH C₁₈ column with methanol and formic acid using isotope labelled internal standards. Analytes were detected in multireaction monitoring mode on a Xevo TQ. Semi‐quantification of vemurafenib metabolites was performed using the same analytical system and sample preparation with gradient elution. The vemurafenib method was successfully validated in the range 0.5–100 μg/mL according to international guidelines. The metabolite method was partially validated owing to the lack of commercially available reference materials. For the first time concentration levels at steady state for melanoma patients treated with vemurafenib is presented. The low abundance of vemurafenib metabolites suggests that they lack clinical significance. Copyright © 2016 John Wiley & Sons, Ltd.     

A simple MALDI target plate with channel design to improve detection sensitivity and reproducibility for quantitative analysis of biomolecules

Overcoming the detrimental effects of sweet spots during crystallization is an important step to improve the quantitative abilities of matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry. In this study, we introduce MALDI targets, which exhibit a channel design to reduce sweet spot phenomena and improve reproducibility. The size of the channels was 3.0 mm in length, 0.35 mm in depth, and 0.40 mm in width, adjusted to the width of the implemented laser beam. For sample deposition, the matrix/sample mixture was homogenously deposited into the channels using capillary action. To demonstrate the proof‐of‐principle, the novel plates were used for the quantification of acetyl‐L‐carnitine in human blood plasma using a combined standard addition and isotope dilution method. The results showed that the reproducibility of acetyl‐L‐carnitine detection was highly improved over a conventional MALDI‐MS assay, with RSD values of less than 5.9% in comparison with 15.6% using the regular MALDI method. The limits of quantification using the new plates were lowered approximately two‐fold in comparison with a standard rastering approach on a smooth stainless‐steel plate. Matrix effects were also assessed and shown to be negligible. The new assay was subsequently applied to the quantification of acetyl‐L‐carnitine in human plasma samples.     

Detection and validated quantification of toxic alkaloids in human blood plasma--comparison of LC-APCI-MS with LC-ESI-MS/MS

Poisonings with toxic plants may occur after abuse, intentional or accidental ingestion of plants. For diagnosis of such poisonings, multianalyte procedures were developed for detection and validated quantification of the toxic alkaloids aconitine, atropine, colchicine, coniine, cytisine, nicotine and its metabolite cotinine, physostigmine, and scopolamine in plasma using LC-APCI-MS and LC-ESI-MS/MS. After mixed-mode solid-phase extraction of 1 ml of plasma, the analytes were separated using a C8 base select separation column and gradient elution (acetonitrile/ammonium formate, pH 3.5). Calibration curves were used for quantification with cotinine-d(3), benzoylecgonine-d(3), and trimipramine-d(3) as internal standards. The method was validated according to international guidelines. Both assays were selective for the tested compounds. No instability was observed after repeated freezing and thawing or in processed samples. The assays were linear for coniine, cytisine, nicotine and its metabolite cotinine, from 50 to 1000 ng/ml using LC-APCI-MS and 1 to 1000 ng/ml using LC-ESI-MS/MS, respectively, and for aconitine, atropine, colchicine, physostigmine, and scopolamine from 5 to 100 ng/ml for LC-APCI-MS and 0.1 to 100 ng/ml for LC-ESI-MS/MS, respectively. Accuracy ranged from -38.6 to 14.0%, repeatability from 2.5 to 13.5%, and intermediate precision from 4.8 to 13.5% using LC-APCI-MS and from -38.3 to 8.3% for accuracy, from 3.5 to 13.8%, for repeatability, and from 4.3 to 14.7% for intermediate precision using LC-ESI-MS/MS. The lower limit of quantification was fixed at the lowest calibrator in the linearity experiments. With the exception of the greater sensitivity and higher identification power, LC-ESI-MS/MS had no major advantages over LC-APCI-MS. Both presented assays were applicable for sensitive detection of all studied analytes and for accurate and precise quantification, with the exception of the rather volatile nicotine. The applicability of the assays was demonstrated by analysis of plasma samples from suspected poisoning cases.