High-throughput and simultaneous analysis of eight central-acting muscle relaxants in human plasma by ultra-performance liquid chromatography–tandem mass spectrometry in the positive and negative ionization modes

In this report, a high-throughput and sensitive method for analysis of eight central-acting muscle relaxants in human plasma by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) in the positive and negative ionization modes using tolbutamide as internal standard is presented. After pretreatment of a plasma sample by solid-phase extraction with an Oasis HLB cartridge, muscle relaxants were analyzed by UPLC with Acquity UPLC BEH C₁₈ column and Acquity TQD tandem quadrupole mass spectrometer equipped with an electrospray ionization interface. The calibration curves for muscle relaxants spiked into human plasma equally showed good linearities in the nanogram per milliliter order range. The detection limits (signal-to-noise ratio = 3) was as low as 0.1–2 ng/mL. The method gave satisfactory recovery rates, accuracy, and precision for quality control samples spiked with muscle relaxants. To further validate the present method, 250 mg of chlorphenesin carbamate was orally administered to a healthy male volunteer, and the concentrations of chlorphenesin carbamate in plasma were measured 0.5, 1, 2, 4, 6, and 8 h after dosing; their concentrations in human plasma were between 0.62 and 2.44 μg/mL. To our knowledge, this is the first report describing simultaneous analysis of over more than two central-acting muscle relaxants by liquid chromatography–tandem mass spectrometry. This has been realized by the capability of our instrument for simultaneous multiple reaction monitoring of the target compounds in both positive and negative ionization modes. Therefore, the present method seems very useful in forensic and clinical toxicology and pharmacokinetic studies.     

Liquid chromatography tandem mass spectrometry determination of total budesonide levels in dog plasma after inhalation exposure

A sensitive and selective method to quantify budesonide in dog plasma samples was developed and fully validated. Liquid–liquid extraction was followed by solid-phase extraction and liquid chromatography–tandem mass spectrometry with electrospray ionization. After reconstitution of the analytes in the mobile phase, samples were analysed by reversed-phase liquid chromatography with isocratic elution. d8-Budesonide was used as an internal standard, and characteristic transitions of d8-budesonide and budesonide were used for quantification. The method was validated with respect to selectivity, specificity, linearity, recovery, repeatability, reproducibility and limits of detection and quantification. The validated method was successfully applied to monitor the plasma levels of budesonide in dogs exposed to clinical doses of inhaled and intravenous drug.     

Direct analysis of free amino acids by mixed‐mode chromatography with tandem mass spectrometry

We developed a straightforward, robust, and relatively fast method for the analysis of amino acids by mixed‐mode high‐performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The method does not involve derivatization and allows the detection of 21 amino acids, representing a wide range of isoelectric points, in less than 40 min. Chromatographic separation was governed by a silica‐based mixed‐mode column providing simultaneous hydrophobic and ion exchange separation mechanisms. The use of tandem mass spectrometry increased selectivity, reducing potential problems associated with poor selectivity in the chromatographic system. For an injection volume of 1 μL, we obtained detection limits <3 μM for the majority of analytes. For all analytes, a linearity of r > 0.99 was obtained, recovery in matrix was >86%, and the retention times were highly reproducible. The method was successfully applied to soil solution and fungal culture samples, demonstrating the advantages in successfully avoiding issues associated with high amounts of substances that may interfere with derivatization‐based methods. This method represents an alternative to derivatization‐based methods and can be applied in areas where sample matrices are highly complex.     

Dried plasma spots as an alternative sample collection technique for the quantitative LC-MS/MS determination of gabapentin

Dried plasma spots were employed as an alternative sample collection technique for the quantitative determination of gabapentin in human plasma, using an automated liquid chromatography tandem mass spectrometry method. After the methanolic extraction of single plasma, 1/8-in. disks which contained only 1.98 μL plasma volume, were placed in 96-well format plates, then gabapentin and its internal standard, 4-aminocyclohexanecarboxylic acid, were derivatized with n-butanol/HCl (3 M) and detected as butyl esters. The assay exhibited excellent linearity over the concentration range of 40.0–10.0 × 10³ ng/mL, which is suitable for the determination of gabapentin after per os administration of a single tablet. Variations in intra- and inter-assay accuracy and precision were within internationally accepted criteria. Homogeneity of plasma spots was proven, whilst butyl ester stability for both analytes was estimated and found very satisfactory. The quantitative analysis of Gabapentin with dried plasma spot specimens seems to be a prominent and advantageous technique, especially when applied to pharmacokinetic studies, where plasma sampling procedure becomes rapid and required plasma volumes negligible.     

LC-MS/MS quantitative analysis of reducing carbohydrates in soil solutions extracted from crop rhizospheres

A simple, sensitive, and specific analytical method has been developed for the quantitative determination of 15 reducing carbohydrates in the soil solution of crop rhizosphere. Reducing carbohydrates were derivatized with 1-phenyl-3-methyl-5-pyrazolone, separated by reversed-phase high-performance liquid chromatography and detected by electrospray ionization tandem mass spectrometry. Lower limits of quantitation of 2 ng/mL were achieved for all carbohydrates. Quantitation was performed using peak area ratios (analyte/internal standard) and a calibration curve spiked in water with glucose-d₂ as the internal standard. Calibration curves showed excellent linearity over the range 2–100 ng/mL (10–1,000 ng/mL for glucose). The method has been tested with quality control samples spiked in water and soil solution samples obtained from the rhizosphere of wheat and canola and has been found to provide accurate and precise results.     

Quantitative Analysis of Triptolide in Human Whole Blood by LC–APCI-IT-MS-MS

In this study, the development and validation of an analytical method for triptolide in whole blood using high-performance liquid chromatography coupled with atmospheric-pressure chemical ionization ion trap tandem mass spectrometry (LC–APCI-IT-MS-MS) is reported. This is the first report of the systematic development and validation of an LC–MS-MS method for the quantitation of triptolide in human whole blood using prednisolone as an internal standard (IS). Prior to LC–MS-MS analysis, liquid–liquid extraction with ethyl acetate was used to isolate them from the biological matrix. Validation parameters such as specificity/selectivity, limit of quantitation (LOQ), linearity, precision, accuracy and stability are evaluated for this method. The calibration curve was linear (r ² = 0.9973) in the concentration range of 0.5–100.0 ng mL⁻¹ in human whole blood with a lower limit of quantitation of 0.5 ng mL⁻¹. Intra- and inter-day relative standard deviations (RSDs) were less than 8.6 and 11.7%, respectively. Extraction recoveries of triptolide ranged from 81.5 to 88.1%. This assay can be used to determine trace triptolide in human whole blood.     

Liquid chromatography-tandem mass spectrometry (LC/APCI-MS/MS) methods for the quantification of captan and folpet phthalimide metabolites in human plasma and urine

Captan and folpet are fungicides largely used in agriculture. They have similar chemical structures, except that folpet has an aromatic ring unlike captan. Their half-lives in blood are very short, given that they are readily broken down to tetrahydrophthalimide (THPI) and phthalimide (PI), respectively. Few authors measured these biomarkers in plasma or urine, and analysis was conducted either by gas chromatography coupled to mass spectrometry or liquid chromatography with UV detection. The objective of this study was thus to develop simple, sensitive and specific liquid chromatography–atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS) methods to quantify both THPI and PI in human plasma and urine. Briefly, deuterated THPI was added as an internal standard and purification was performed by solid-phase extraction followed by LC/APCI-MS/MS analysis in negative ion mode for both compounds. Validation of the methods was conducted using spiked blank plasma and urine samples at concentrations ranging from 1 to 250 μg/L and 1 to 50 μg/L, respectively, along with samples of volunteers and workers exposed to captan or folpet. The methods showed a good linearity (R ² > 0.99), recovery (on average 90% for THPI and 75% for PI), intra- and inter-day precision (RSD, <15%) and accuracy (<20%), and stability. The limit of detection was 0.58 μg/L in urine and 1.47 μg/L in plasma for THPI and 1.14 and 2.17 μg/L, respectively, for PI. The described methods proved to be accurate and suitable to determine the toxicokinetics of both metabolites in human plasma and urine.     

Measuring multiple neurochemicals and related metabolites in blood and brain of the rhesus monkey by using dual microdialysis sampling and capillary hydrophilic interaction chromatography-mass spectrometry

In vivo measurement of multiple functionally related neurochemicals and metabolites (NMs) is highly interesting but remains challenging in the field of basic neuroscience and clinical research. We present here an analytical method for determining five functionally and metabolically related polar substances, including acetylcholine (quaternary ammonium), lactate and pyruvate (organic acids), as well as glutamine and glutamate (amino acids). These NMs are acquired from samples of the brain and the blood of non-human primates in parallel by dual microdialysis, and subsequently analyzed by a direct capillary hydrophilic interaction chromatography (HILIC)–mass spectrometry (MS) based method. To obtain high sensitivity in electrospray ionization (ESI)–MS, lactate and pyruvate were detected in negative ionization mode whereas the other NMs were detected in positive ionization mode during each HILIC-MS run. The method was validated for linearity, the limits of detection and quantification, precision, accuracy, stability and matrix effect. The detection limit of acetylcholine, lactate, pyruvate, glutamine, and glutamate was 150 pM, 3 μM, 2 μM, 5 nM, and 50 nM, respectively. This allowed us to quantitatively and simultaneously measure the concentrations of all the substances from the acquired dialysates. The concentration ratios of both lactate/pyruvate and glutamine/glutamate were found to be higher in the brain compared to blood (p < 0.05). The reliable and simultaneous quantification of these five NMs from brain and blood samples allows us to investigate their relative distribution in the brain and blood, and most importantly paves the way for future non-invasive studies of the functional and metabolic relation of these substances to each other.     

Plasma lipid analysis by hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A novel method for the analysis of endogenous lipids and related compounds was developed employing hydrophilic interaction liquid chromatography with electrospray ionization tandem mass spectrometry. A hydrophilic interaction liquid chromatography with carbamoyl stationary phase achieved clear separation of phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, ceramide, and mono‐hexsosyl ceramide groups with good peak area repeatability (RSD% < 10) and linearity (R² > 0.99). The established method was applied to human plasma assays and a total of 117 endogenous lipids were successfully detected and reproducibly identified. In addition, we investigated the simultaneous detection of small polar metabolites such as amino and organic acids co‐existing in the same biological samples processed in a single analytical run with lipids. Our results show that hydrophilic interaction liquid chromatography is a useful tool for human plasma lipidome analysis and offers more comprehensive metabolome coverage.     

Determination of tandospirone in human plasma by a liquid chromatography–tandem mass spectrometry method

A rapid, sensitive, and selective liquid chromatography–tandem mass spectrometry method for the detection of tandospirone in human plasma is described. It was employed in a pharmacokinetic study. The analyte and internal standard diphenhydramine were extracted from plasma using liquid–liquid extraction, then separated on a Zorbax XDB C₁₈ column using a mobile phase of methanol–water–formic acid (80:20:0.5, v/v/v). The detection was performed with a tandem mass spectrometer equipped with an electrospray ionization source. Linearity was established in the concentration range of 10.0-5,000 pg/ml. The lower limit of quantification was 10.0 pg/ml. The intraday and interday relative standard deviation across three validation runs over the entire concentration range was less than 13%. Accuracy determined at three concentrations (25.0, 200, and 4,000 pg/ml for tandospirone) ranged from 94.4 to 102.1%. Each plasma sample was chromatographed within 3.4 min. The method proved to be highly selective and suitable for bioequivalence evaluation of different formulations containing tandospirone and clinical pharmacokinetic investigation of tandospirone.     

Quantitative estimation of riluzole in human plasma by LC-ESI-MS/MS and its application to a bioequivalence study

A novel simple, sensitive, selective, and rapid high-performance liquid chromatography coupled with tandem mass spectrometry method was developed and validated for quantification of riluzole in human plasma. The chromatography was performed by using a Zorbax-SB-C18 (4.6 × 75 mm, 3.5 μm) column , isocratic mobile phase 0.1% formic acid/acetonitrile (10:90 v/v), and an isotope-labeled internal standard (IS), [¹³C,¹⁵N₂]riluzole. The extraction of drug and internal standard was performed by liquid–liquid extraction and analyzed by MS in the multiple reaction monitoring (MRM) mode using the respective [M+H]⁺ ions, m/z 235.0/165.9 for riluzole and m/z 238.1/169.0 for the IS. The calibration curve was linear over the concentration range 0.5–500.0 ng/ml for riluzole in human plasma. The limit of quantification (LOQ) was demonstrated at 0.5 ng/ml. The within-batch and between-batch precision were 0.6–2.3% and 1.4–5.7%, and accuracy was 97.1–101.1% and 98.8–101.2% for riluzole respectively. Drug and IS were eluted within 3.0 min. The validated method was successfully applied in a bioequivalence study of riluzole in human plasma.     

Distinct hepatic lipid profile of hypertriglyceridemic mice determined by easy ambient sonic-spray ionization mass spectrometry

Easy ambient sonic-spray ionization mass spectrometry (EASI-MS) was used to interrogate the hepatic lipid profiles of hypertriglyceridemic and control normotriglyceridemic mice. The analyses of ex vivo complex lipid mixtures were made directly with EASI-MS without accompanying separation steps. Intense ions for phosphatidylcholines and triacylglycerols were observed in the positive ion mode whereas the spectra in the negative ion mode provided profiles of phosphatidylethanolamines and phosphatidylinositol. EASI-MS was coupled to high-performance thin-layer chromatography for analysis of free fatty acids. Fourier transform–ion cyclotron resonance–mass spectrometry was also employed to confirm the identity of the detected lipids. We demonstrated higher incorporation of oleic acid in phosphatidylcholine and triacylglycerol composition, higher relative abundance of arachidonic acid containing phosphatidylinositol, and overall distinct free fatty acid profile in the livers of genetic hypertriglyceridemic mice. We propose that these alterations in liver lipid composition are related to the higher tissue and body metabolic rates described in these hypertriglyceridemic mice.     

Characterization of binding and bioaccessibility of Cr in Cr-enriched yeast by sequential extraction followed by two-dimensional liquid chromatography with mass spectrometric detection

Sequential extraction (water, Driselase, protease XIV) and extraction with simulated gastric and intestinal fluids were proposed to characterize the binding and the bioaccessibility of chromium in two commercial food supplements obtained by incorporation of this element into yeast. Chromium in Cr-enriched yeast was found to be hardly extractable with water, Driselase, or simulated gastric fluid (recoveries of approximately 10–20%), but proteolysis or gastrointestinal fluid digestion released more than half of the chromium present. Fractionation with size-exclusion chromatography with Cr-specific detection by inductively coupled plasma mass spectrometry (ICP MS) allowed the distinction of two fractions: one below approximately 1 kDa and one 1–5 kDa; they contained the entirety of the released Cr with proportions varying as a function of the extracting solution and the origin of sample. When collected and investigated by reversed-phase high-performance liquid chromatography–ICP MS, the low molecular mass fraction was found to release Cr(III), whereas the heavier one showed most of Cr bound in fairly stable hydrophobic complexes. However, an attempt of their identification by electrospray ionization MS/MS and matrix-assisted laser desorption ionization MS was not successful.      

Automated mass spectrometric analysis of urinary and plasma serotonin

Serotonin emerges as crucial neurotransmitter and hormone in a growing number of different physiologic processes. Besides extensive serotonin production previously noted in patients with metastatic carcinoid tumors, serotonin now is implicated in liver cell regeneration and bone formation. The aim was to develop a rapid, sensitive, and highly selective automated on-line solid-phase extraction method coupled to high-performance liquid chromatography–tandem mass spectrometry (XLC-MS/MS) to quantify low serotonin concentrations in matrices such as platelet-poor plasma and urine. Fifty microliters plasma or 2.5 μL urine equivalent were pre-purified by automated on-line solid-phase extraction, using weak cation exchange. Chromatography of serotonin and its deuterated internal standard was performed with hydrophilic interaction chromatography. Mass spectrometric detection was operated in multiple reaction monitoring mode using a quadrupole tandem mass spectrometer with positive electrospray ionization. Serotonin concentrations were determined in platelet-poor plasma of metastatic carcinoid patients (n = 23) and healthy controls (n = 22). Urinary reference intervals were set by analyzing 24-h urine collections of 120 healthy subjects. Total run-time was 6 min. Intra- and inter-assay analytical variation were <10%. Linearity in the 0–7300 μmol/L calibration range was excellent (R² > 0.99). Quantification limits were 30 and 0.9 nmol/L in urine and plasma, respectively. Platelet-poor serotonin concentrations in metastatic carcinoid patients were significantly higher than in controls. The urinary reference interval was 10–78 μmol/mol creatinine. Serotonin analysis with sensitive and specific XLC-MS/MS overcomes limitations of conventional HPLC. This enables accurate quantification of serotonin for both routine diagnostic procedures and research in serotonin-related disorders.     

Development of a liquid chromatography-tandem mass spectrometry method for plasma-free metanephrines with ion-pairing turbulent flow online extraction

Liquid chromatography–tandem mass spectrometry has become the preferred technology to measure unconjugated metanephrine and normetanephrine in plasma because of its high sensitivity and specificity over immunoassay and gas chromatography–mass spectrometry. In our earlier study, plasma metanephrines were extracted with offline ion-pairing solid-phase extraction and quantified by liquid chromatography–tandem mass spectrometry with porous graphitic carbon column based chromatography. In this study, we aim to automate the sample preparation with turbulent flow online extraction technology and maintain or improve the analytical performance previously achieved from the offline approach. The online extraction was done with a mixed-mode cation exchange turbulent flow chromatography column assisted with ion-pairing reagent and porous graphitic column was used for chromatographic separation. The total online extraction and analytical LC runtime was 12 min. This method was linear from 6.3 to 455.4 pg/mL for metanephrine; 12.6 to 954.5 pg/mL for normetanephrine with an accuracy of 80.6% to 93.5% and 80.9% to 101.7%, respectively. The lower limit of quantitation was 6.3 pg/mL for metanephrine and 12.6 pg/mL for normetanephrine. Inter-assay and intra-assay precision for metanephrine and normetanephrine at low and high concentration levels ranged from 2.0% to 10.5%. In conclusion, we have developed a fast and sensitive automated online turbulent flow extraction method for the quantitative analysis of plasma metanephrines. Ion-pairing reagent was necessary for the success of this method.     

Quantitative determination of Phakellistatin 13, a new cyclic heptapeptide,in rat plasma by liquid chromatography/tandem mass spectrometry: application to a pharmacokinetic study

A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, followed by a 96-well protein precipitation, has been developed and fully validated for the determination of Phakellistatin 13 (PK13), a new cyclic heptapeptide isolated from the sponge Phakellia fusca Thiele, in rat plasma. After protein precipitation of the plasma samples (50 μL) in a 96-well plate by methanol (200 μL) containing the internal standard Pseudostellarin B (20 ng/mL), the plate was vortex mixed for 3 min. Following filtration for 5 min, the filtrate was directly injected into the LC-MS/MS system. The analytes were separated on an XB-C18 analytical column (5 μm, 50 mm × 4.6 mm i.d.) using an eluent of methanol–water (85:15, v/v) and detected by electrospray ionization mass spectrometry in the negative multiple reaction monitoring mode with a chromatographic run time of 5.0 min. The method was sensitive with a lower limit of quantification (LLOQ) of 0.1 ng/mL, with good linearity (r > 0.999) over the quantitation range of 0.1–5 ng/mL. The validation results demonstrated that this method was significantly specific, accurate, precise, and was successfully applied in measuring levels of PK13 in rat plasma following intravenous administration of 20, 50, and 100 μg/kg of peptide in rats, respectively, which was suitable for the preclinical pharmacokinetic studies on PK13.     

Determination of lipid mediators in breast cancer cells using lyophilization‒supercritical fluid extraction online coupled with supercritical fluid chromatography‒quadrupole tandem mass spectrometry

A lyophilization?supercritical fluid extraction coupled with supercritical fluid chromatography?quadrupole tandem mass spectrometry online method was developed for the determination of lipid mediators in breast cancer cells. Supercritical fluid extraction was applied to the cell samples for the first time due to the use of lyophilization. The conditions of supercritical fluid extraction and supercritical fluid chromatography?quadrupole tandem mass spectrometry were investigated systematically. Under the optimized conditions, all the calibration curves for the lipid mediators showed good linearity (correlation coefficient > 0.99). The limits of detection and the limits of quantification were in the range of 0.190?5.36 pg and 0.560?16.2 pg, respectively. The recoveries were in the range of 70.3?125%. The relative standard deviations of the precision ranged from 1.49?18.7% and the accuracies were higher than 84%. Compared with liquid?liquid extraction coupled with liquid chromatography and tandem mass spectrometry method, the present approach reduced the manual labor and obtained higher sensitivity as well as higher extraction recoveries for all 15 lipid mediators. Finally, the online method was applied to the quantification of lipid mediators in breast cancer cells and normal mammary epithelial cells. On the basis of the results, this lyophilization?supercritical fluid extraction online coupled with supercritical fluid chromatography?quadrupole tandem mass spectrometry method showed great promise in the analysis of lipid mediators in complex biological samples.     

Simultaneous quantification of fatty acids in serum with dual derivatization by liquid chromatography-tandem mass spectrometry

Fatty acids have multitudinous biological functions and play a crucial role in many biological processes, but due to poor ionization efficiency and lack of appropriate internal standards, the comprehensive quantification of fatty acids by liquid chromatography-tandem mass spectrometry is still challenging. In this study, a new, accurate, and reliable method for quantifying 30 fatty acids in serum using dual derivatization was proposed. Indole-3-acetic acid hydrazide derivants of fatty acids were used as the internal standard and indole-3-carboxylic acid hydrazide derivants of them were used to quantify. The derivatization conditions were systematically optimized and the method validation results showed good linearity with R² > 0.9942, low detection limit (0.03–0.6 nM), precision (1.6%–9.8% for intra-day and 4.6%–14.1% for inter-day), recovery (88.2%–107.2% with relative standard deviation < 10.5%), matrix effect (88.3%–105.2% with the relative standard deviation < 9.9%) and stability (3.4%–13.8% for fatty acids derivants in 24 h at 4°C and 4.2%–13.8% for three freeze-thaw cycles). Finally, this method was successfully applied to quantify fatty acids in serum samples of Alzheimer's patients. In contrast to the healthy control group, nine fatty acids showed a significant increase in the Alzheimer's disease group.     

Determination of 30 Free Fatty Acids in Two Famous Tibetan Medicines by HPLC with Fluorescence Detection and Mass Spectrometric Identification

A simple and sensitive high-performance liquid chromatographic (HPLC) method with fluorescence detection and mass spectrometric identification has been developed for analysis of 30 long-chain and short-chain free fatty acids (FFAs). The fatty acids were derivatized to their esters with 1-[2-(p-toluenesulfonate)ethyl]-2-phenylimidazole-[4,5-f]-9,10-phenanthrene (TSPP) in N,N-dimethylformamide (DMF) at 90 °C with anhydrous K₂CO₃ as catalyst. A mixture of C₁–C₃₀ fatty acids was completely separated within 60 min by gradient elution on a reversed-phase C₈ column. Qualitative identification of the acids was performed by atmospheric-pressure chemical ionization mass spectrometry (APCI–MS) in positive-ion mode. The fluorescence excitation and emission wavelengths were 260 and 380 nm, respectively. Quantitative determination of the 30 acids in two Tibetan medicines Gentiana straminea and G. dahurica was performed. The results indicated that the medicines contained many FFAs. Linear correlation coefficients for the FFA derivatives were >0.9991. Relative standard deviations (RSDs, n = 6) for the fatty acid derivatives were <3%. Detection limits (at a signal-to-noise ratio of 3:1) were 3.1–38 fmol. When the fatty acid derivatives were determined in the two real samples results were satisfactory and the sensitivity and reproducibility of the method were good.     

A sensitive and efficient procedure for the high throughput determination of banned aromatic amines in textiles and leather products aided by advanced sample composition

We have developed and validated a quantitative liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI MS/MS) procedure for the simultaneous determination of seven natural and semisynthetic tropane alkaloids in plasma: atropine (d-hyoscyamine/l-hyoscyamine), cocaine, homatropine, ipratropium, littorine, N-butylscopolamine, and scopolamine. Plasma and serum samples were precipitated for deproteinization (recovery 88–94%), followed by reversed-phase-based liquid chromatography prior to positive electrospray ionization for detection by multiple reaction monitoring using a linear ion trap quadrupole mass spectrometer. All analytes were quantified using cocaine-d3 as an internal standard suitable and reliable for robust, precise (coefficient of variation 2–13%), and accurate (87–122%) measurement within a linear range of 3 orders of magnitude (0.05–50 ng/ml plasma). The method was exemplarily applied to stability studies in phosphate-buffered saline, human serum, and rabbit serum. Each alkaloid was incubated separately and samples were taken at distinct incubation time points. Supernatants of diverse alkaloids at corresponding time points were pooled and subjected to simultaneous LC-ESI MS/MS quantification. This combinatorial analysis design allowed us to analyze the stability of samples with a drastically reduced number of chromatographic runs. In the presence of rabbit serum, all tropane alkaloids tested were degraded significantly within minutes to hours, with the exception of the stable semisynthetic compounds ipratropium and N-butylscopolamine. In contrast, in the presence of equal concentrations of human serum, no degradation was observed for any of the compounds, with the exception of cocaine. Relevant enzymes involved in enzymatic degradation are discussed.