Liquid Chromatographic Mass Spectrometric Method Research Articles

Liquid chromatography-mass spectrometric method for the simultaneous analysis of branched-chain amino acids and their ketoacids from dried blood spot as secondary analytes for the detection of maple syrup urine disease

BackgroundMaple syrup urine disease (MSUD) is an aminoacidopathy caused by a defective branched-chain alpha-ketoacid dehydrogenase complex, leading to the accumulation of branched-chain amino acids (BCAAs) and their respective keto acids (BCKAs). A comprehensive test was developed to measure BCAAs and BCKAs using LC-MS from dried blood spot (DBS) samples for the diagnosis and prevention of MSUD in newborns and infants. MethodsAnalytes were extracted from DBS using a methanol:0.1 % v/v formic acid solution (75:25) containing internal standards and analyzed on a Luna PFP column (150 mm × 4.6 mm, 3 µm) at a flow rate of 0.3 mL/min. The method was validated for linearity, accuracy, precision, recovery, carry-over, matrix effect, hematocrit, blood volume, and punch position effects. Biomarker stability in the matrix and stock solution was assessed. Correlation with the plasma method was determined using Pearson’s correlation coefficient and Bland-Altman analysis. The method established reference ranges for the Udupi district population in South India. ResultsThe method demonstrated linearity (r2 > 0.99), with a lower limit of detection at 2 µM (BCAA) and 1 µM (BCKA), and acceptable recovery of QC samples. Hematocrit, blood volume, punch position, and storage condition effects were within acceptable limits. Correlation and Bland-Altman analysis showed strong interconvertibility between plasma and DBS assays. Reference ranges for leucine, isoleucine, valine, KIC, KIV, and KMV were established. ConclusionThe developed DBS method, requiring no derivatization and involving simple sample preparation with short run times, is a cost-effective and reliable approach for the confirmatory diagnosis of MSUD.

Liquid Chromatography Mass Spectrometric Method and a Fluorometric 96 Well Plate Assay for Determination of Thiamine in Salmonid Eggs.

Thiamine deficiency is a large contributor to reduced reproduction success among salmonids throughout the northern hemisphere. In Scandinavia, this reproduction disorder is known as M74; while in North America, it is known as early mortality syndrome (EMS). The disorder fluctuates in magnitude from year to year. During years with high prevalence of the disorder, salmonid hatcheries that stock various aquatic systems to maintain the population size experience difficulties filling their quotas without thiamine treatment of alevins. The disorder is monitored both by observing the survival rate and by measuring the thiamine content of prefertilized eggs in the hatcheries. Here, a simple extraction procedure is presented, which allows for quantitative determination of the various phosphorylated forms of thiamine using liquid chromatography mass spectrometry but also allows for extraction in 96 deep-well plates and measurement of the total thiamine content using fluorescence monitoring with a plate reader, following oxidation of thiamine to thiochrome. The latter procedure could also be integrated into a highly portal system where the thiochrome is determined using the DeNovix QFX analyzer. The newly developed extraction procedure and cleanup method for fluorescence measurement represent the most versatile and simple methods to date for monitoring of thiamine in salmonid eggs. The methods produced accurate and precise data with quantification limits below the limit where the deficiency causes 100% lethality.

Targeted screening and profiling of massive components of colistimethate sodium by two-dimensional-liquid chromatography-mass spectrometry based on self-constructed compound database

In-depth study of the components of polymyxins is the key to controlling the quality of this class of antibiotics. Similarities and variations of components present significant analytical challenges. A two-dimensional (2D) liquid chromatography-mass spectrometric (LC-MS) method was established for screening and comprehensive profiling of compositions of the antibiotic colistimethate sodium (CMS). A high concentration of phosphate buffer mobile phase was used in the first-dimensional LC system to get the components well separated. For efficient and high-accuracy screening of CMS, a targeted method based on a self-constructed high resolution mass spectrum database of CMS components was established. The database was built based on the commercial MassHunter Personal Compound Database and Library (PCDL) software and its accuracy of the compound matching result was verified with 6 known components before being applied to genuine sample screening. On this basis, the unknown peaks in the CMS chromatograms were deduced and assigned. The molecular formula, group composition and origins of a total of 99 compounds, of which the combined area percentage accounted for more than 95% of CMS components, were deduced by this 2D-LC-MS method combined with the MassHunter PCDL. This profiling method was highly efficient and could distinguish hundreds of components within 3 h, providing reliable results for quality control of this kind of complex drugs.

Quantification of asciminib by LC-MS/MS method in human plasma: Validation and stability studies

An accurate, sensitive, and linear liquid chromatographic tandem mass spectrometric method was developed for the quantification of asciminib in human plasma. Voriconazole, internal standard (IS), and asciminib were isolated from the plasma samples by C18-cartridges. Chromatographic isolation was processed with Atlantis/dC18 (2.1 × 100 mm, 3 μm) stationary phase and a mobile solvent system of formic acid (0.1%) and methyl alcohol (20:80, v:v), conveyed at a 0.7 ml/minute flowing rate. Analytes were quantified by ionization in a positive approach with electrospray ionization at the mass transitions of (m/z): asciminib, 450.11/239.09, and voriconazole (IS), 350.3/281.1. No interference by constituents of plasma blank or other components was detected. The association between asciminib concentration levels and their respective peak response fractions to voriconazole was rectilinear between 39.0 and 1,586.0 ng/ml. Intraday and interday precisions were ≤4.79% for asciminib. The interday bias was between −4.28% and 5.76%, and the intraday bias was −3.75% and 4.53%. The mean measured extraction recovery of asciminib was 99.06%. The recovery of IS was 98.34%. Asciminib was subjected to long-term, bench-top, freeze-thaw, short-term stability, dry extract, auto-sampling, and stock solution stability at Low quality control and High quality control levels, and it was stable at all these conditions. The established technique can be utilized for the regular quantification of asciminib in plasma samples in industries, forensic laboratories, and clinical research organizations.

Pharmacokinetics of Moxidectin combined with Albendazole or Albendazole plus Diethylcarbamazine for Bancroftian Filariasis.

Moxidectin (MOX) is a milbemycin endectocide recently approved by the U.S. FDA for the treatment of onchocerciasis in persons at least 12 years of age. MOX has been shown to have a good safety profile in recent clinical trials. The efficacy of MOX for the treatment of lymphatic filariasis (LF) and its potential use in mass drug administration protocols for the elimination of LF is currently under evaluation. In the context of a clinical trial, we investigated the pharmacokinetics and drug interactions of a combination of MOX plus albendazole (ALB) with or without diethylcarbamazine (DEC) compared to ivermectin (IVM) plus ALB with or without DEC in the following four different treatment arms: (I) IVM (0.2mg/kg) plus DEC (6 mg/kg) and ALB (400mg); (II) IVM plus ALB; (III) MOX (8 mg) plus DEC and ALB; and (IV) MOX plus ALB. Drug concentrations were determined using validated liquid chromatography-mass spectrometric methods. Pharmacokinetic parameters were determined using standard non-compartmental analysis methods. Statistical analysis was performed using JMP software. Fifty-eight of 164 study participants (53 men and five women) were included with ages ranging from 18 to 63 yrs (mean = 37). MOX apparent oral clearance (Cl/F) ranged from 0.7 to 10.8 L/hr with Cmax values ranging from 20.8 to 314.5 ng/mL. The mean (range) area under the curve (AUC)0-∞ for MOX, 3405 ng*hr/mL (742-11376), and IVM 1906 ng*hr/mL (692-5900), varied over a ~15.3 and ~8.5-fold range, respectively. The geometric mean ratio for Cmax, AUC0-t, and AUC0-∞ were within the no-drug interaction range of 80-125% for all drugs. This indicates that the addition of MOX to ALB alone or ALB plus DEC for LF therapy did not alter the drug exposure of co-administered drugs compared to IVM combinations. Clinical Trial Registration: NCT04410406, https://clinicaltrials.gov/.

Screening for chemicals with thyroid hormone-disrupting effects using zebrafish embryo

Thyroid disruption is an increasingly recognized issue in the use and development of chemicals and new drugs, especially to help toxicologist to complement the reproductive and developmental toxicology information of chemicals. Still, adequate assessment methods are scarce and often suffer a trade-off between physiological relevance and labor- and cost-intensive assays. Here, we present a tiered approach for a medium-throughput screening of chemicals to identify their thyroid disrupting potential in zebrafish embryos as a New Approach Methodology (NAM). After identifying the maximum tolerated concentrations, we exposed zebrafish larvae to sub-adverse effect levels of the reference compounds benzophenone-2, bisphenol A, phenylthiourea, potassium perchlorate, propylthiouracil, and phloroglucinol to exclude any systemic toxicity. Applying the transgenic zebrafish line that carries a gene for the red fluorescence protein (Tg(tg:mCherry)) under the thyroglobulin promoter, we could identify the thyroid disrupting effects of the chemicals by a time and cost-effective image analysis measuring the fluorescence levels in the thyroid glands. Our observations could be confirmed by altered expression patterns of genes involved in the hypothalamus-pituitary-thyroid (HPT) axis. Finally, to anchor the observed thyroid disruption, we determined some changes in the Thyroid hormone levels of triiodothyronine (T3) and Thyroxine (T4) using a newly developed liquid chromatography mass spectrometric (LCMS) method. The presented approach carries the potential to extend the toolbox for legislative authorities and chemical producers for the assessment of thyroid-specific endocrine disruption and to overcome current challenges in the evaluation of endocrine disruptors.

Development and validation of a method based on liquid chromatography-mass spectrometry for comprehensive profiling of phenolic compounds in rice

The consumption of rice grains containing phytochemicals associated with antioxidant activity can potentially reduce the risk of chronic diseases. These phytochemicals, which include phenolic acids, anthoxanthins, and anthocyanins, are present in both free and bound forms in rice grains. However, there is limited documentation on the development of a method for determining the simultaneous amount of both free and bound phenolic compounds using sensitive analytical techniques. To address this gap, a study was conducted to develop and validate a liquid chromatography-mass spectrometric detection method for quantifying eighteen phenolic compounds in rice grains, both in free and bound forms. Paracetamol was used as the internal standard to minimize the effect of matrix interferences. The method was validated by measuring its working range, linearity, accuracy, precision, limits for detection and quantification, robustness, and expanded uncertainty. The method has a wide working range of 1.0 to 30.0 mg/kg for each phenolic compound and good linearity, with regression coefficients of calibration graphs greater than 0.999. Accuracy was determined using the recoveries after fortification at three concentration levels, which were in the range of 83.1 to 101.2%. Precision was expressed as a percentage of relative standard deviation (%RSD), which was below 7.62% for all determined phenolic compounds. The detection limits (LoD) and quantification limits (LoQ) ranged between 0.53 and 0.75 mg/kg and 0.54–0.77 mg/kg, respectively. The percentages of expanded uncertainties at the coverage factor of 2 (k = 2) were below 11.7%. The method was also evaluated for its robustness by obtaining recoveries at satisfactory levels for three different kinds of cereal; wheat, maize, and millet. The validated method is an accurate and reproducible approach for the simultaneous determination of phenolic compounds present at low levels in a complex matrix such as rice. The study also found that the phenolic profiles vary among rice genotypes, which can significantly impact human health by varying the nutraceutical properties of rice grains as health-promoting aspects.

A Sensitive Liquid Chromatography-Mass Spectrometric Method for Determination of Bisoprolol in Rat Serum after Pre-Column Derivatization.

Two derivatization regents were reacted with bisoprolol (BIS), followed by liquid-chromatography-mass spectrometric analysis. 3-Bromomethyl-propyphenazone (BMP) and dansyl-chloride (Dns-Cl) were reacted via the secondary amino group using a catalyst to accelerate the reaction progress and completeness with minimal reaction byproducts. The sensitivity and the ionization efficiency of both BIS-methyl-propyphenazone (BIS-MP) and BIS-Dns via electrospray ionization were studied. The sensitivity of BIS-MP was superior to BIS-Dns. The derivatization procedure, extraction procedure, and LC-MS method were optimized and validated to achieve the monitoring of BIS in rat serum at a picogram scale. The calibration curve showed a regression coefficient value of 0.999 within a concentration spanning a range of 10-500pg/mL. The detection limit and quantitation were 4 and 10pg/mL, respectively. The intraday and inter-day precision values (% relative standard deviation) ranged from 0.53 to 6.91%, whereas the accuracy values (expressed as % error) ranged from -4.20 to -0.77%. The pharmacokinetic parameters were: 15,280pg/mL for a maximum concentration of BIS (Cmax) at the maximum time (Tmax) of 1 h. BIS's elimination half-life (t1/2) was determined to be 3 h. The value of the area under the concentration-time curve (AUC0 - t) was 34,370pg/mLh.

Development of a comprehensive two-dimensional liquid chromatographic mass spectrometric method for the non-targeted identification of poly- and perfluoroalkyl substances in aqueous film-forming foams

In this research, we developed an online comprehensive two-dimensional liquid chromatographic (LC × LC) method hyphenated with high-resolution mass spectrometry (HRMS) for the non-targeted identification of poly- and perfluorinated compounds (PFASs) in fire-fighting aqueous-film forming foams (AFFFs). The method exploited the combination of mixed-mode weak anion exchange-reversed phase with a octadecyl stationary phase, separating PFASs according to ionic classes and chain length.To develop and optimize the LC × LC method we used a reference training set of twenty-four anionic PFASs, representing the main classes of compounds occurring in AFFFs and covering a wide range of physicochemical properties. In particular, we investigated different modulation approaches to reduce injection band broadening and breakthrough in the second dimension separation. Active solvent and stationary phase assisted modulations were compared, with the best results obtained with the last approach. In the optimal conditions, the predicted peak capacity corrected for undersampling was higher than three-hundred in a separation space of about 60 min. Subsequently, the developed method was applied to the non-targeted analysis of two AFFF samples for the identification of homologous series of PFASs, in which it was possible to identify up to thirty-nine potential compounds of interest utilizing Kendrick mass defect analysis. Even within the samples, the features considered potential PFAS by mass defect analysis elute in the chromatographic regions discriminating for the ionic group and/or the chain length, thus confirming the applicability of the method presented for the analysis of AFFF mixtures and, to a further extent, of environmental matrices affected by the AFFF.

Analysis of cycloheximide in rat specimens using liquid chromatography with tandem mass spectrometry detection

The development of a selective and sensitive high-performance liquid chromatographic tandem mass spectrometric method for the determination of cycloheximide (CHX) in rat blood and plasma is described. The extraction of CHX and colchicine as internal standard from blood fluid (0.1 mL) was achieved using n-hexane: dichloromethane: isopropanol (20:10:1 v/v/v). The mobile phase, a combination of methanol:10 mM ammonium acetate (85:15, v/v), was pumped at 0.2 mL/min through a C18 analytical column with a run time of 3.5 min. Detection was carried out by electrospray positive ionization mass spectrometry in the multiple-reaction monitoring (MRM) mode. The assay exhibited excellent linearity (r2 > 0.999) in peak area response over the concentration ranges of 2–1000 ng CHX /mL blood fluid. The mean absolute recoveries for 20, 100 and 500 ng/mL CHX in blood fluid using the present extraction procedure were > 97%. The intra- and inter-day coefficients of variation in the plasma and blood and mean error were < 13% at different concentrations. Samples had limited stability at room temperature, and speedy processing is needed. After intravenous administration, rats had measurable concentrations of CHX for up to 24 h after dosing with 1 mg/kg of cycloheximide. The method displayed a high caliber of sensitivity and selectivity for detecting very low concentrations of CHX in rats.

Rapid and Stable Liquid Chromatographic Tandem Mass Spectrometric Method for Simultaneous Estimation of Pioglitazone, Keto Pioglitazone and Hydroxy Pioglitazone in Human Plasma: Application to Bioequivalence Study

High performance liquid chromatographic tandem mass spectrometric method for the determination of pioglitazone, keto pioglitazone (M-III) and hydroxy pioglitazone (M-IV) in human plasma has been developed and validated using pioglitazone-D4, keto pioglitazone-D4 and hydroxy pioglitazone-D5 as internal standards. Solid phase extraction was carried out for sample preparation to extract analyte and internal standard from human plasma. The extracted sample was injected through autosampler in HPLC connected with Hypersil Gold, 100 mm × 4.6 mm, 5 μm using mobile phase consisting of methanol:solution A:solution B - 80:10:10 v/v/v). Pioglitazone, keto pioglitazone (M-III) and hydroxy pioglitazone (M-IV) were chromatographically separated and detected using the MS detector. The best-fit lines using weighting factor (1/concentration2) linear least square regression analysis were obtained by peak area ratio of pioglitazone, keto pioglitazone (M-III) and hydroxy pioglitazone (M-IV) with their internal standards pioglitazone-D4, keto pioglitazone-D4 and hydroxy pioglitazone-D5, respectively. This report provides the results of various validation parameters including stability studies and extended precision and accuracy which is required for long study samples batch analysis. This analytical method is valid for the determination of pioglitazone in the range of 18.9 ng/mL to 2994.4 ng/mL), keto pioglitazone (M-III) (3.23 ng/mL to 512.60 ng/mL), hydroxy pioglitazone (M-IV) 10.1 ng/mL to 1603.8 ng/mL) and using pioglitazone-D4, keto pioglitazone-D4, hydroxy pioglitazone-D5, respectively as internal standards in human plasma using a Hypersil Gold, 100 mm × 4.6 mm, 5 μm column.

Liquid chromatography-mass spectrometric methods for trace quantification of potential genotoxic impurities in ivacaftor and lumacaftor

The objective of the current study was to develop and validate the sensitive LC-MS methods for trace analysis of genotoxic impurities in Ivacaftor and Lumacaftor. The first method is for the trace analysis of 2,4-di-tert-butyl-5-nitrophenol in ivacaftor and the second method is for the trace analysis of 1-(2,2-difluoro-1,3-benzodioxol-5yl)-cyclopropane carboxylic acid and 3-carboxyphenyl boronic acid in lumacaftor.High pure analytical grade solvents and reagents were used for this study. The chromatographic separation was performed on Luna C18 (250×4.6mm, 5.0μm) at a column temperature of 25°C using eluent consisting of acetonitrile and 0.1% v/v formic acid in water in a gradient elution mode. The eluent was run at a flow of 1.0mL/min and injection volume of 20μL.The linearity, precision and accuracy of the developed methods was validated over the concentration range of 0.35-15.0ppm for 2,4-di-tert-butyl-5-nitrophenol, 0.30-15.0ppm for 1-(2,2-difluoro-1,3-benzodioxol-5yl)-cyclopropane carboxylic acid and 0.23-15.0ppm for 3-carboxyphenyl boronic acid. In both methods, interference was not observed at the retention time of analyte peaks. All the analytes were found to be stable in solution for a period of 48h.The proposed methods are reliable, sensitive, precise, accurate, and robust for the trace level quantification of genotoxic impurities in Ivacaftor and Lumacaftor. These methods can be successfully implemented in the quality control lab for routine analysis.

Quantification for Antibody-Conjugated Drug in Trastuzumab Emtansine and Application to In Vitro Linker Stability and In Vivo Pharmacokinetic Study in Rat Using an Immuno-Affinity Capture Liquid Chromatography-Mass Spectrometric Method

Trastuzumab emtansine (T-DM1, brand name: Kadcyla®) is the first FDA-approved antibody-drug conjugate (ADC) for metastatic human epidermal growth factor receptor 2 positive (HER2+) breast cancer. It consists of three components: trastuzumab, an anti-HER2 monoclonal antibody, maytansinoid (DM1) as a cytotoxic drug, and maleimidomethyl cyclohexane-1-carboxylate (MCC) as a linker. In particular, the MCC linker is known as a non-cleavable linker and has a feature of being conjugated to DM1 by a covalent thioether bond. In this study, we developed an immuno-affinity capture liquid chromatography-mass spectrometric (LC-MS/MS) assay for quantifying the antibody-conjugated drug (acDrug) component of T-DM1. To quantify acDrug, desulfurated DM1 was prepared using a chemical desulfuration pretreatment and quantified as an acDrug. A quadratic regression (weighted 1/concentration), with equation y = ax2 + bx + c, was used to fit the calibration curves over the concentration range of 17.09~1709.44 ng/mL for the acDrug of T-DM1. The quantification run met the in-house acceptance criteria of ±25% accuracy and precision values for the quality control (QC) samples. In conclusion, an immuno-affinity capture LC-MS/MS assay was successfully developed to quantify acDrug of T-DM1 and applied to evaluate in vitro plasma linker stability and preclinical pharmacokinetic (PK) study in rats. This assay could be helpful when applied to other ADCs with the same linker-cytotoxic drug platform.

UPLC-MS/MS Method Validation for Estimation of Resveratrol in Rat Skin from Liposphere Gel Formulation and Its Application to Dermatokinetic Studies in Rats.

For the quantification of resveratrol (RV) in the Wistar rat skin, an ultra-performance liquid chromatography-mass spectrometric (UPLC-MS/MS) method was developed and validated on ACQUITY UPLC BEH C18 column (1.7 μm). The mobile phase ratio of methanol (A) and 2% formic acid (B) (ratio 10: 90% v/v, 80: 20 v/v) at isocratic elution with flow rate 0.3 mL/min, and run time 3 min was used for analysis. In addition, the use of multiple reaction monitoring (MRM)/ES+ mode to detect the analytes and to track parents to daughter ion transition of 229.17 > 107.04 m/z (time scan 3 min, retention time 1.48) for RV and curcumin as an internal standard shows 369.16 > 176.93m/z (scan time is 2.80 min, retention time is 1.11), respectively. Linearity was observed in the range of 2.5 to 2,000 ng/mL (R2 = 0.987). Precision and accuracy on rat skin were within the acceptability range (RE%: ±15; RSD%: ±15). Moreover, it showed a good percentage recovery and found within acceptance limit 90-110%. Lower limit of detection and quantitation for the method observed to be 2.5 and 20 ng.mL-1, respectively. Method application indicated successful determination of dermatokinetics parameters of RV from lipospheres gel and suspension in the rats.

Simultaneous determination of ezetimibe, atorvastatin and simvastatin using quadrupole LC-MS: Application to combined tablets and plasma after SPE

AbstractA simple and sensitive liquid chromatography-mass spectrometric (LC-MS) method has been developed and validated for the simultaneous determination of ezetimibe (EZE), atorvastatin calcium (ATO), and simvastatin (SMV) in combined dosage forms and human plasma. Successful separation of the studied drugs was achieved on a Zorbax Eclipse Plus C18 column (3.0 × 150 mm, 5 µm) using a mobile phase consisting of acetonitrile and 0.1% formic acid in water (65:35, v/v) at a flow rate of 0.5 mL min−1. Total run time was 9.3 min and diclofenac sodium was used as internal standard (IS). Positive selected ion monitoring (SIM) mode was applied where, the monitored ions were those at m/z values of 392.1, 559.3, 296.0, and 441.4 corresponding to EZE, ATO, IS, and SMV, respectively. The method was fully validated according to the ICH guidelines. The intraday and interday precision showed relative SD values not more than 1.77 and 1.99%; respectively. The limits of detection (LOD) were 0.25, 0.25, and 0.75 ng mL−1 while the limits of quantification (LOQ) were 1.25, 0.75, and 2.5 ng mL−1 for EZE, ATO, and SMV, respectively. The developed method was applied on two types of combined tablets concerning drug assay with mean percent recoveries within acceptable range. The method has been extended to the determination of the studied drugs in human plasma where, a solid phase extraction method was optimized for their extraction with percent recovery not less than 97%.

Follow up: palmitic acid ester of tetrahydrocannabinol (THC) and palmitic acid diester of 11-hydroxy-THC- unsuccessful search for additional THC metabolites.

In a previous investigation we searched for the occurrence of palmitic acid ester compounds of delta9-tetrahydrocannabinol (THC) and its primary metabolite 11-hydroxy-delta9-THC (11-OH-THC) in human body fluids and tissues (THC palmitic acid monoester [THC-Pal] and 11-OH-THC palmitic acid diester [11-OH-THC-DiPal]). As those esters could not be detected in various human body fluids (e.g. blood) or tissues (e.g. adipose tissue) we extended the investigation analyzing adipose tissue samples of mice previously given synthetic THC or a cannabis extract. In total, 48 adipose tissue samples previously tested positive for THC by means of a liquid chromatographic triple quadrupole mass spectrometric (LC-QQQ-MS) method were analyzed for the presence of THC-Pal and 11-OH-THC-DiPal by means of LC-QQQ-MS. THC-Pal and 11-OH-THC-DiPal were not detected among the adipose tissue samples analyzed despite the presence of high THC concentrations within the adipose tissue. THC concentrations in adipose tissue were in a range of approximately 7-2,595ng/g (median: 468ng/g, average: 704ng/g). A (site-specific) synthesis of 11-OH-THC palmitic acid monoesters (11-hydroxy-delta9-THC-1-palmitate and 11-palmitoyloxy-delta9-THC) still remains to be done. After synthesis of these monoesters, their presence in the body fluids and tissues after THC administration should be investigated.

Follow up: palmitic acid ester of tetrahydrocannabinol (THC) and palmitic acid diester of 11-hydroxy-THC- unsuccessful search for additional THC metabolites.

In a previous investigation we searched for the occurrence of palmitic acid ester compounds of delta9-tetrahydrocannabinol (THC) and its primary metabolite 11-hydroxy-delta9-THC (11-OH-THC) in human body fluids and tissues (THC palmitic acid monoester [THC-Pal] and 11-OH-THC palmitic acid diester [11-OH-THC-DiPal]). As those esters could not be detected in various human body fluids (e.g. blood) or tissues (e.g. adipose tissue) we extended the investigation analyzing adipose tissue samples of mice previously given synthetic THC or a cannabis extract. In total, 48 adipose tissue samples previously tested positive for THC by means of a liquid chromatographic triple quadrupole mass spectrometric (LC-QQQ-MS) method were analyzed for the presence of THC-Pal and 11-OH-THC-DiPal by means of LC-QQQ-MS. THC-Pal and 11-OH-THC-DiPal were not detected among the adipose tissue samples analyzed despite the presence of high THC concentrations within the adipose tissue. THC concentrations in adipose tissue were in a range of approximately 7-2,595ng/g (median: 468ng/g, average: 704ng/g). A (site-specific) synthesis of 11-OH-THC palmitic acid monoesters (11-hydroxy-delta9-THC-1-palmitate and 11-palmitoyloxy-delta9-THC) still remains to be done. After synthesis of these monoesters, their presence in the body fluids and tissues after THC administration should be investigated.

Development and validation of a liquid chromatographic tandem mass spectrometric method for the analysis of patulin in apple and apple juice.

This study reports a robust and sensitive method for rapid testing of patulin in apple and apple juice. The method involved extraction of homogenised samples (10g) with ethyl acetate (10mL) and clean up by dispersive-solid phase extraction using primary secondary amine (25mg/mL). Prior to the LC-MS/MS analysis, the cleaned extract was reconstituted in methanol/water (2:8). The optimised LC-MS condition provided a symmetric peak of patulin within a short LC-runtime of 5min. The recoveries at the limit of quantification (0.005mg/kg) and higher levels were satisfactory (> 80%), with the precision-RSDr (< 11%). In an inter-laboratory comparison study involving 13 accredited laboratories, the reproducibility-RSDR and HorRat values ranged between 4.80 and 6.08% and between 0.18 and 0.23 respectively, indicating a satisfactory method-precision. The z-scores of the participating laboratories were within ± 2. When the method was applied to incurred samples, the contamination range was 0.008-0.225mg/kg and 0.018-0.034mg/kg for apple and juice respectively, demonstrating a satisfactory performance in terms of precision. Based on the solvent standard, matrix-matched standard and standard-addition approaches, the calibration graphs provided similar quantitative performances. Because of its reliability, robustness and time-effectiveness, the method can be recommended for regulatory testing purposes.

Method Development and Validation of Liquid Chromatography-Mass Spectrometric Method (LC-MS) for the Simultaneous Determination of Darunavir in Human Plasma

Method Development and Validation of Liquid Chromatography-Mass Spectrometric Method (LC-MS) for the Simultaneous Determination of Darunavir in Human Plasma

Quantification of selected pharmaceutical compounds in water using liquid chromatography-electrospray ionisation mass spectrometry (LC-ESI-MS)

The detection and quantitation of pharmaceutical compounds (PCs) in different environmental matrices is still a challenge, due to their extremely low (ng-μg) concentrations and the lack of rapid and sensitive analytical techniques. A number of techniques, such as enzyme-linked immunosorbent assay (ELISA), chromatography, electrophoresis, and electrochemical methods have been explored. These methods are limited by their poor sensitivity. In this study, a hyphenated liquid chromatography-mass spectrometric (LC-MS) method was developed, validated, and tested for the detection and quantification of seven active pharmaceutical compounds, with solid-phase extraction for analytes recovery and separation of interference from the aqueous matrix. The sensitivity achieved for the method allowed for LODs (μg/L) of 0.0439, 0.0684, 0.1219, 0.0710, 0.1129, 0.0447, 0.0837 and LOQs (μg/L) of 0.1462, 0.2281, 0.4065, 0.2367, 0.3763, 0.1492, 0.2792, for lamivudine, acetaminophen, vancomycin, ciprofloxacin, sulfamethoxazole, diclofenac, and ivermectin, respectively, within a linear range of 0.01–0.1 μg/mL. Other ICH validation parameters are also discussed. The different PCs were positive in 61 % of the tested surface waters, with diclofenac present only in two of the sampling points. The concentrations at which they occurred were variable and ranged between ND and 398.98 μg/L.