QC Samples Research Articles

Development of efficient on-bead protein elution process coupled to ultra-high performance liquid chromatography–tandem mass spectrometry to determine immunoglobulin G subclass and glycosylation for discovery of bio-signatures in pancreatic disease

Type 1 autoimmune pancreatitis (AIP) is a kind of IgG4-related disease in which higher IgG4 and total IgG levels have been found in patient serum. Due to the similar imaging features and laboratory parameters between AIP and pancreatic ductal adenocarcinoma (PDAC), a differential diagnosis is still challenging. Since IgG profiles can be potential bio-signatures for disease, we developed and validated a method which coupled on-bead enzymatic protein elution process to an efficient UHPLC–MS/MS method to determine IgG subclass and glycosylation. A stable-isotope labeled IgG was incorporated as internal standard to achieve accurate quantification. For calibration curves, the correlation coefficients for total IgG and the four IgG subclasses were higher than 0.995. Intraday (n = 5) and interday (n = 3) precisions of the peak area ratios of LLOQ, low, medium, and high QC samples were all less than 6.6% relative standard deviation (% RSD), and the accuracies were between 93.5 and 114.9%. Calibration curves, precision, and accuracy were also evaluated for 26 IgG glycopeptides. The method was applied to samples from healthy controls and patients with AIP and PDAC. Distinct IgG patterns were discovered among the groups, and 7 glycopeptides showed high potential in differentiating AIP and PDAC. The results demonstrated that the developed method is suitable for multi-feature analysis of human IgG, and the discovered IgG profiles can be used as bio-signatures for AIP and PDAC.

The First Comprehensive LC-MS/MS Method Allowing Dissection of the Thiamine Pathway in Plants.

Arabidopsis thaliana serves as a model plant for genetic research, including vitamin research. When aiming at engineering the thiamine (vitamin B1) pathway in plants, the availability of tools that allow the quantitative determination of different intermediates in the biosynthesis pathway is of pivotal importance. This is a challenge, given the nature of the compounds and the minute quantities of genetically engineered material that may be available for analysis. Here, we report on the first LC-MS/MS method for the simultaneous quantification of thiamine, its mono- and diphosphate derivatives and its precursors 4-methyl-5-(2-hydroxyethyl) thiazole (HET) and 4-amino-2-methyl-5-hydroxymethylpyrimidine (HMP). This method was optimized and validated for the quantitative determination of these analytes in Arabidopsis thaliana. All analytes were chromatographically separated within less than 2.5 min during an 8 min run. No unacceptable interferences were found. The method was fully validated based on international guidelines. Accuracy (%bias) and total imprecision (%CV) were within preset acceptance criteria for all analytes in both QC and real samples. All analytes were stable in extracted samples when stored for 48 h at 4 °C (autosampler stability) and when reanalyzed after storage at -80 °C and -20 °C for 2 weeks (freeze/thaw stability). We demonstrated the start material should be stored at -80 °C to ensure stability of all analytes during short- and long-term storage (up to 3 months). The validity and applicability of the developed procedure was demonstrated via its successful application on Arabidopsis lines, genetically engineered to enhance thiamine content.

GMP manufacture of Regulatory T cells (Tregs) to facilitated reduced reliance on immunosuppressive drugs following renal transplantation

Seetha Abdul Wahab, Matthew Brook, Giovanna Lombardi, Joanna Hester, Paul Harden, Fadi Issa
 Due to significant side effects, it is desirable to be able to reduce patient’s dependency on immunosuppressive drugs after organ transplantation. Personalised Treg therapy offers a promising route to facilitating a reduction in immunosuppression. The TWO Study employs a GMP-compliant manufacturing process to produce autologous Tregs for therapy. GMP is a quality management system that entails production and quality control of advanced therapy medicinal products (ATMPs) ensuring that ATMPs are consistently produced per clinical trial authorisation or product specification. This work is undertaken at a purpose-built GMP facility at Guy’s Hospital, London. Patient samples of peripheral blood are received from Oxford and then volume reduced aseptically to 40 mls using Sepax. CD8+cells are eliminated using CliniMACS CD8 reagent. CliniMACS CD25 reagent is then added to isolate CD25+ cells. Isolated cells are expanded using Expact Treg bead suspension kit and Rapamycin. Cells are fed every two days and subsequently re-stimulated twice at five feeding cycle intervals or until the required cell number is reached. The cells are then pooled and centrifuged at 1000xg. The resulting pellet is re-suspended and the Expact expansion beads removed using CliniMACS tubing set LS. Finally, the cell product is formulated in CryoStor CS-10 to a maximum dose concentration of 10x106 cells/kg patient body weight. At the same time, a back-up dose is prepared as well as reference and QC samples. These are all cooled to -80oC at c.1oC/min and stored in liquid nitrogen vapour phase until dispatch. As of end 2019, three patient samples have been processed. Tregs were purified and propagated to 194.4, 282.1, and 183.4-fold expansion yielding 3.5x109, 2.2x109, and 3.1x109 cells respectively. Samples 1 and 2 have been released for treatment after phenotyping and testing for suppression activity, sterility, and contamination with aerobic/anaerobic pathogens and mycoplasma. The third product is scheduled to be released for treatment in May 2020.

An Ultrasensitive LC-APPI-MS/MS Method for Simultaneous Determination of Ciclesonide and Active Metabolite Desisobutyryl-Ciclesonide in Human Serum and Its Application to a Clinical Study.

The development of more efficient drug delivery devices for ciclesonide inhalation products requires an ultrasensitive bioanalytical method to measure systematic exposure of ciclesonide (CIC) and its active metabolite desisobutyryl-ciclesonide (des-CIC) in humans. Serum sample was extracted with 1-chlorobutane. A reversed-phase liquid chromatography coupled with atmospheric pressure photoionization-tandem mass spectrometry (LC-APPI-MS/MS) method was used for quantification of 1-500 pg/mL for both analytes in a 0.500-mL serum. The analysis time was 4.7 min/injection. CIC-d11 and des-CIC-d11 were used as the internal standards. Calibration curves showed good linearity (r2 > 0.99) for both analytes. This novel method was precise and accurate with interassay precision and accuracy of all within 9.6% CV and ± 4.0% bias for regular QC samples. Extraction recovery was approximately 85% for both analytes. Serum samples are stable for 3 freeze-thaw cycles, 24 h at bench top, and up to 706 days at both -20 °C and -70 °C. This method was successfully used to support a pharmacokinetic (PK) comparison between the inhalation suspensions and an inhalation aerosol of ciclesonide in healthy participants. The method robustness was also supported by the good incurred sample reanalysis reproducibility. APPI, a highly selective and sensitive ionization source, made possible for quantifying CIC and des-CIC with a lower limit of quantification (LLOQ) of 1 pg/mL in human serum by LC-MS/MS. A 10-fold sensitivity improvement from the most sensitive reported method (LLOQ, 10 pg/mL) is essential to fully characterize the PK profiles of CIC and des-CIC in support of the clinical development of the ciclesonide-related medications for patients.

Quantitative Determination of Loratadine in Rat Plasma by LC–MS/MS Method and Its Application in a Bioavailability Study

A sensitive, simple and fast LC–MS/MS method was established and validated for the determination of loratadine in rat plasma. The preparation of plasma sample was completed through a one-step liquid–liquid extraction method. An UHPLC column Phenomenex Kinetex C18 (50 mm × 2.1 mm, 2.6 µm) was utilized to achieve the chromatography separation. Mobile phases A and B were acetonitrile and water with a ratio of 45:55 (v/v) under an isocratic gradient, both comprising 0.2% formic acid. Multiple reaction monitoring mode (MRM) was carried out on the triple quadrupole detection in position ionization mode with an electrospray ionization source, the transitions of m/z 383.1 → 337.2 and m/z 284.9 → 193.3 were used to determine loratadine and diazepam (internal standard), respectively. The total analysis run time was 3.5 min and the elution time of loratadine and diazepam occurred at 1.1 and 2.1 min, respectively. This method has the advantages of higher sensitivity (0.04 ng mL−1), wider linear range (0.04–400 ng mL−1, r2 > 0.995), shorter analysis time (3.5 min), needing only a smaller plasma volume (50 μL) and simple preparation process. The accuracy and precision of loratadine in all QC samples met the requirements. This novel method was successfully applied to a bioavailability research of loratadine nanosuspensions in rats.

Rapid profiling method for the analysis of lipids in human plasma using ion mobility enabled-reversed phase-ultra high performance liquid chromatography/mass spectrometry

The incorporation of ion mobility (IM) into LC–MS analysis has been demonstrated to result in the generation of superior quality MS and MS/MS spectral data as well as providing enhanced resolution in the IM dimension based on lipid class. Here a sub 4 min microbore LC-ion mobility-accurate mass MS (LC-IM-MS) method has been developed for the rapid, profiling of lipids in biological fluids. The method was scaled directly from a conventional, 12 min, LC-MS analysis maintaining the chromatographic performance and lipid separation observed in the longer methodology giving a 75% saving in mobile phase consumption and analysis time. Because of the additional dimension of separation provided by IM, improvements in mass spectral quality from the increased resolution of co-eluting species were also seen when compared to the same separation without IM, thus aiding the identification of target lipids. When applied to human plasma samples some 5037 (positive ESI) and 2020 (negative ESI) mass/retention time features were detected following adduct deconvolution and, of these, 3727 and 800 of those present in the pooled plasma QC samples had a CV of below 30% for positive and negative ESI modes respectively. The method was applied to the analysis of a pilot set of commercially sourced breast cancer plasma samples enabling the differentiation of samples from healthy controls and patients based on their lipid phenotypes. Analysis of the resulting data showed that phosphatidylcholines, triglycerides and diglycerides exhibited lower expression and phosphatidylserine showed increased expression in the breast cancer samples compared to those of healthy subjects. The coefficients of variation, determined by reference to the QC data, for all of the features identified as potential markers of disease, were 6% or less.

P1.11-11 Initial Discovery of Exhaled Small Polar Energetics-Related Metabolites by GC-MS for Lung Cancer Risk Assessment

There is a need for non-invasive airway-based biomarkers in lung carcinogenesis for both risk assessment, and earlier diagnosis. Exhaled breath condensate (EBC) contains airway lining fluid molecules, including small molecules of polar and non-polar lipid origin, presumably in part from epithelial cellular origins. Here we pilot a GC-MS strategy for measurement of small, polar molecules in exhaled breath condensate in EBC from lung cancer patients and controls. Exhaled breath condensate (EBC) was collected non-invasively, using a handheld device (RTube) in ambulatory subjects engaged/recruited/consented through our pulmonary and thoracic surgery practices, under IRB protocol. A volume of 50 ul of EBC samples were combined with 200uL of methanol, containing 2 internal standards, 1nmol U13_succinate, 5nmol U13_citrate. Then, the samples were vortexed and 240 μl of supernatant was transferred to a sampling vial. The samples were dried under gentle nitrogen flow and derivatized with a two-step derivatization procedure, including a methoxyamine step for 90 minutes, and a silylation step for 60 minutes. QC sample was run multiple times during the analysis. The samples were analyzed by gas chromatography time-of-flight mass spectrometry (GC-TOFMS premier, Waters, USA). A number of 282 variables were detected after alignment and excluding any known artificial peaks, 49 of them were annotated. The data was normalized to the intensity of the sum of all the metabolites. The data set was then imported into SIMCA-p software (Umeå, Sweden) for multivariate analysis. A multivariate case-control ROC discriminant analysis compared the clinical model (AUC 0.72) to clinical plus exhaled small polar discriminant metabolites combined (AUC 0.92), showed incremental discrimination attributable to these metabolites (p=2.22e-62). ROC comparing clinical model (AUC 0.72) to clinical model plus metabolites (AUC 0.92); the difference attributable to metabolites was significant (p=2.22e-62). This exhaled biomarker platform can yield case-control discriminant small polar molecule sets related to known metabolic pathways, some of which are known to be deranged in cancers. Once further distilled and validated, our goal is to apply this non-invasive biomarker approach to prospective cohorts for non-invasive lung cancer risk assessment of the at-risk epithelium, in order to better select higher risk individuals to undergo effective CT screening. Supported by NIH-R21 CA192168-01; DoD-CDMRP- LC150738, NIH-NCI P60DK020541.

A sensitive and reliable RP-HPLC method for the detection of cimetidine – a H2 receptor antagonist in human plasma

Bioanalytical methods for bioequivalence studies require high sensibility and rapidity due to the large number of samples and the low plasma concentration of drugs. The present study aimed to develop and validate a high-performance liquid chromatography method to quantify cimetidine (CMT) in human plasma and to apply it in a bioequivalence study. Spiked plasma of 500 µl (l, m and h concentration) was used for the assay. The HPLC injection volume was 20μl of the reconstitute sample where, 2 ml of ethyl acetate used for extraction purposes. Cimetidine was prepared separately for low (80 ng/ml), medium (2000 ng/ml) and high (3600 ng/ml) concentrations and internal standard (ranitidine) concentration was 3000 ng/ml. Freeze thawing and long terms stability were conducted at -25º c. The individual calibration curve for spiked standards was linear with R2= 0.99. The inaccuracy values for QC samples were within 15% of the actual value and not more than 20% for the LOQ. The limit of quantitation (LOQ) was 40 ng/ml, which was also the lowest concentration of cimetidine that was quantitated with the variability of 5.9%. The within day precision and between day precision for LOQ were 10.8 and 5.9 respectively. The retention time for the analyte was 4.1-4.5 minutes during the within a day and between day results. The mean % inaccuracy values for low, medium and high concentration were 6.8, 5.6 and 7.8 respectively for within day and 2.4, 6.1 and 7.9 respectively for between days. The within day and between day % inaccuracy for LOQ concentration was 12.4 and 5.5 respectively. The mean recoveries for low, medium and high concentration of cimetidine were 80.2, 70.9 and 74.2. The overall mean recovery for cimetidine was 75.1%. The maximum inaccuracy for freeze thaw cycle and long term stability samples for low, medium and high was found with CV less than 15% for all concentrations, indicating that cimetidine is stable. The developed method was precise and accurate and was suitable to be applied for the bioequivalence study of cimetidine.

The determination of human peripheral blood mononuclear cell counts using a genomic DNA standard and application in tenofovir diphosphate quantitation

A fluorescent quantitation method to determine PBMC-derived DNA amounts using purified human genomic DNA (gDNA) as the reference standard was developed and validated. gDNA was measured in a fluorescence-based assay using a DNA intercalant, SYBR green. The fluorescence signal was proportional to the amount (mass) of DNA in the sample.The results confirmed a linear fit from 0.0665 to 1.17 μg/μL for gDNA, corresponding to 2.0 × 106 to 35.0 × 106 cells/PBMC sample. Intra-batch and inter-batch accuracy (%RE) was within ±15%, and precision (%CV) was <15%. Benchtop stability, freeze/thaw stability and long term storage stability of gDNA in QC sample matrix, PBMC pellets samples, and pellet debris samples, respectively, as well as dilution linearity had been established. Consistency between hemocytometry cell counting method and gDNA-based counting method was established. 6 out of 6 evaluated PBMC lots had hemocytometry cell counts that were within ±20% of the cell counts determined by the gDNA method.This method was used in conjunction with a validated LC-MS/MS method to determine the level of tenofovir diphosphate (TFV-DP), the active intracellular metabolite of the prodrugs tenofovir alafenamide (TAF) and tenofovir disoproxil fumarate (TDF), measured in PBMCs in clinical trials of TAF or TDF-containing fixed dose combinations.

LC-MS/MS method with chemical derivatization for quantitation of L-threonate in human plasma.

An LC-MS/MS-based bioanalytical method has been developed to measure the concentration of L-threonate at its endogenous level in human plasma. Following isotope dilution and protein precipitation, the samples were acetylated and chromatographed under reversed-phase conditions for baseline separation of the derivatized L-threonate and its stereoisomer D-erythronate. The method was assessed by a fit-for-purpose validation with a calibration range from 100 to 10,000 ng/mL. The intra-run coefficients of variation (CVs) were <3.6% and the inter-run CV was 3.2% for the QC samples at endogenous level. At the lower limit of quantitation, the intra-run CV was 6.1% and the average inaccuracy was -1.4%. This method provides an efficient and reliable quantitation of L-threonate and could be useful to certain biomarker investigators.

Development and validation of novel LC–MS/MS method for determination of Lusutrombopag in rat plasma and its application to pharmacokinetic studies

A novel, simple and sensitive method for the determination of Lusutrombopag in rat plasma using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was developed and validated. The determination was performed on an API4000 triple quadrupole mass spectrometry in the multiple reaction monitoring mode using the respective [M+H]+ ions m/z 593.1 → 272.3 for Lusutrombopag. The limit of detection was 0.5 ng/mL, and the lower limit of quantification was 2.0 ng/mL in rat plasma. Good linearity was obtained over the range of 2.0–150.0 ng/mL and the correlation coefficient was found to be 0.9998. The intra and inter-day precisions were found to be 3.8–6.9% and 6.8–10.5%, respectively. The intra and inter-day accuracy derived from QC samples was found to be 2.5–4.9% and 5.5–7.2%, respectively. The analyte was stable under various conditions (at room temperature, during freeze-thaw, in the autosampler and under deep-freeze conditions). The F-test and t-test at 95% confidence level were subjected on data for statistical analysis. The developed method was successfully applied to the pharmacokinetic study in rats.

Paclitaxel-Loaded Silk Fibroin Nanoparticles: Method Validation by UHPLC-MS/MS to Assess an Exogenous Approach to Load Cytotoxic Drugs.

The aim of this work was to load an anticancer drug, paclitaxel (PTX), on Silk Fibroin Nanoparticles (SFNs) by using an exogenous approach. SFNs were produced, freeze-dried and then loaded with PTX. An exogenous method allowed us to reduce both drug loss and environmental impact. In order to quantify PTX loaded in SFNs, a simple and reliable method using reversed phase liquid chromatography coupled to tandem mass spectrometry (rp-UHPLC-MS/MS) was developed. This methodology was validated by the determination of spiked QC samples in three consecutive days. Good accuracy and precision of the method were obtained, while the intra-day and inter-day precisions were less than 10.3%. For PTX, the limit of quantitation (LOQ) was 5.0 ng/mL. Recovery from the matrix (SFNs-PTX pellets) was calculated (81.2% at LOQ value) as PTX was entrapped in a new matrix like the polymer silk fibroin-based. This method was successfully applied to determine the encapsulation efficiency (1.00 ± 0.19%) and the nanoparticle loading (0.12 ± 0.02% w/w). The in vitro anticancer activity of SFNs-PTX was tested against CFPAC-1 cancer cells; results demonstrated a very high cytotoxic activity of SFNs-PTX, with a dose dependent inhibition of CFPAC-1 proliferation, confirmed by the IC50 value of 3450 ± 750 ng/mL.

Liquid-phase microextraction in 96-well plates - calibration and accurate quantification of pharmaceuticals in human plasma samples

The use of miniaturized systems with the possibility for automation has become increasingly popular in the field of bioanalysis. As a new approach to liquid phase microextraction in the 96-well format, parallel artificial liquid membrane extraction (PALME) was introduced in 2013. In the present work, the reliability of the quantitative data obtained with PALME was thoroughly evaluated. Amitriptyline, nortriptyline, quetiapine, venlafaxine, o-desmethylvenlafaxine, and fluoxetine were selected as model analytes. The analytes were extracted under non-exhaustive conditions from human plasma samples and the extracts were analyzed directly by LC-MS/MS. Accuracy was within ±15% and precision was <15% when the QC samples were prepared in both pooled plasma and in plasma from multiple sources. Accuracy and precision was superior when stable isotopically labelled (SIL) internal standards were used, as compared to structural analogue internal standards in the plasma samples from multiple sources. SIL internal standards are therefore recommended as the first choice. Assessment of accuracy and precision was also carried out with four different operators performing the extraction procedure, providing accuracy within ±15% and precision <15%. The extraction recoveries were in the range from 48 to 85 %, and non-exhaustive extraction of the analytes did not affect the accuracy and precision of the method. With the method described, up to 96 samples can be extracted with a total extraction time of 60 min and with a total consumption of organic solvent less than 0.4 mL for the whole wellplate. PALME is therefore a new approach to high-throughput sample preparation, providing accurate quantification, along with simple workflow, low consumption of organic solvent, and extensive sample clean-up.

Simultaneous profiling of vitamin D metabolites in serum by supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS)

Simultaneous profiling of serum vitamin D (VD) metabolites with similar structures is a big challenge. Thus, we developed and validated a SFC-MS/MS method, which is capable of eluting hydrophobic molecules, for quantification of VD2/VD3, 25-OH VD2/VD3, 3-epi-25-OH VD2/VD3, 1,25-(OH)2 VD2/VD3 and 24,25-(OH)2 VD2/VD3. VD metabolites were extracted from human serum using acetonitrile solvent. Column stationary phase, elution gradients, flow rate, column temperature, ion-source type and buffer system in post-column make-up solvent were optimized. Baseline separation of 10 VD metabolites can be achieved using PFP column within 10 min; and detection performed under positive electrospray ionization mode allowed quantification of VD metabolites in serum matrix with a limit of quantification (LOQ) varrying from 0.071 to 0.704 ng/mL. The accuracy was controlled with relative bias lower than 5.5% for QC and NIST samples. The developed method showed excellent intra-assay (0.52–7.93% RSD) and inter-assay (1.35–9.04% RSD) precision. The methodology shows enhanced efficiency and sensitivity as compared to LC-MS/MS method using the same column and mass spectrometer, along with significant correlation and low mean difference bias on measurements. For analysis of trace 1,25-(OH)2 VD2 and 1,25-(OH)2 VD3 in normal human serum or plasma, further improvement of LOQ (like derivatization) should be considered. In conclusion, the use of supercritical fluid not only enhanced safety with reduced solvent cost, but also improved retention and sensitivity as compared to LC-MS/MS method. The developed SFC-MS/MS method is appropriate for high throughput analysis of multiple VD metabolites in human serum with reduced solvent and economic cost.

Validation according to European and American regulatory agencies guidelines of an LC-MS/MS method for the quantification of free and total ropivacaine in human plasma.

Background Ropivacaine is a widely used local anaesthetic drug, highly bound to plasma proteins with a free plasma fraction of about 5%. Therefore, the monitoring of free drug concentration is most relevant to perform pharmacokinetic studies and to understand the drug pharmacokinetic/pharmacodynamic (PK/PD) relationship. Methods A high-sensitivity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using reverse-phase LC and electrospray ionisation mass spectrometry with multiple reaction monitoring (MRM) is described for the quantitation of both free and total ropivacaine in human plasma. Ropivacaine-d7 was used as an internal standard (IS). Results The method was validated in the range 0.5-3000 ng/mL, with five levels of QC samples and according to the European Medicine Agency and Food and Drug Administration guidelines. The performance of the method was excellent with a precision in the range 6.2%-14.7%, an accuracy between 93.6% and 113.7% and a coefficient of variation (CV) of the IS-normalised matrix factor below 15%. This suitability of the method for the quantification of free and total ropivacaine in clinical samples was demonstrated with the analysis of samples from patients undergoing knee arthroplasty and receiving a local ropivacaine infiltration. Conclusions A method was developed and validated for the quantification of free and total ropivacaine in human plasma and was shown suitable for the analysis of clinical samples.

Fast and Simple LC-MS/MS Method for Rifampicin Quantification in Human Plasma.

A simple, fast, and cost-effective LC-MS/MS method for quantification of rifampicin in human plasma was developed and fully validated. The plasma samples containing rifampicin and isotopically labelled internal standard rifampicin D8, were cleaned up using a Captiva ND Lipids filtration plate. Chromatographic separation was achieved on an 1290 Infinity liquid chromatograph coupled to 6460 Triple Quadrupole operated in positive mode on a core-shell Kinetex C18 column (50 × 2.1 mm, 2.6 μm) by gradient elution using 0.1% formic acid in water and acetonitrile as a mobile phase. The proposed method is the fastest method published by now, both in terms of sample preparation (approximately one minute per sample) and chromatographic analysis (total run time 2.4 min). Another key benefit is the outstanding sensitivity and wide analytical range (5-40000 μg/L) with good linearity, accuracy, and precision. The method showed almost complete recovery (92%) and absence of any significant matrix effect as demonstrated by uniform responses from QC samples prepared in blood plasma from 6 volunteers (RSD <5%). The proposed method was successfully applied to rifampicin quantification in 340 patients' plasma samples, thus demonstrating its suitability for both therapeutic drug monitoring and pharmacokinetic analysis.

Development and validation of an UPLC-MS/MS method for the therapeutic drug monitoring of oral anti-hormonal drugs in oncology

A liquid chromatography-mass spectrometry assay was developed and validated for simultaneous quantification of anti-hormonal compounds abiraterone, anastrozole, bicalutamide, Δ(4)-abiraterone (D4A), N-desmethyl enzalutamide, enzalutamide, Z-endoxifen, exemestane and letrozole for the purpose of therapeutic drug monitoring (TDM). Plasma samples were prepared with protein precipitation. Analyses were performed with a triple quadrupole mass spectrometer operating in the positive and negative ion-mode. The validated assay ranges from 2 to 200 ng/mL for abiraterone, 0.2–20 ng/mL for D4A, 10–200 ng/mL for anastrozole and letrozole, 1–20 ng/mL for Z-endoxifen, 1.88–37.5 ng/mL for exemestane and 1500–30,000 ng/mL for enzalutamide, N-desmethyl enzalutamide and bicalutamide. Due to low sensitivity for exemestane, the final extract of exemestane patient samples should be concentrated prior to injection and a larger sample volume should be prepared for exemestane patient samples and QC samples to obtain adequate sensitivity. Furthermore, we observed a batch-dependent stability for abiraterone in plasma at room temperature and therefore samples should be shipped on ice. This newly validated method has been successfully applied for routine TDM of anti-hormonal drugs in cancer patients.

Determination of residual glyphosate and aminomethylphosphonic acid in wheat flour and oats samples by non-derivatization method based on ultra-performance liquid chromatography-high resolution mass spectrometry

A rapid and accurate analysis method based on ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) was developed for the determination of residual glyphosate (GLY) and aminomethylphosphonic acid (AMPA) in wheat flour and oats samples. The wheat flour and oats samples were firstly subjected to vortex and ultrasound extraction; then, the extract solution was purified by MCX solid-phase extraction (SPE) cartridges as well as protein precipitation using acetonitrile. The chromatographic separation was carried out on a Dikma Polyamino HILIC column (150 mm×2.0 mm, 5 μm) by linear gradient elution procedure using 5 mmol/L ammonium acetate in water (pH=10.5) and acetonitrile as the elution solvent. An electrospray ion source in negative mode and parallel reaction monitor (PRM) mode was used for quantification by the internal standard method. The instrument conditions for liquid chromatography (LC) and HRMS, and the sample pretreatment conditions for GLY and its metabolite AMPA were systematically optimized. In addition, the matrix effect and injection system residue were investigated, and a comparison of different analytical methods was carried out. The results indicated that GLY and AMPA showed good linearities in the range of 5.0-100.0 μg/L with coefficients (R2) higher than 0.999. The limits of detection (LODs) were found to be 0.005 and 0.05 mg/kg for GLY and AMPA, respectively. The recoveries of GLY and AMPA in the wheat flour and oats samples were in the range of 93.8%-115% and 89.8%-110% at the spiked levels of 0.1, 0.5, and 2.0 mg/kg, respectively, while the relative standard deviations (RSDs) were all less than 10%. The results of the matrix effect test revealed that the matrix inhibition effect could be reduced by using an isotopic internal standard with the matrix effect parameter |η|<3%. Moreover, the injection system residue was effectively controlled with a residual level of less than 1.0%. A comparison of the developed method with the reported derivatization method indicated little difference, with RSDs of 2.19% and 3.07% to the assigned value, respectively. The established analytical method was used for the Food Analysis Performance Assessment Scheme (FAPAS) proficiency test (No. 09122, GLY in oats), and the results were satisfactory with a z value of 0.2. Moreover, the result obtained using this method was very closed to the assigned value of the FAPAS QC sample, with a recovery of 102.2% (No. T09119QC, GLY in wheat flour). The proposed method is fast, simple, sensitive, and accurate, and it can be applied for the daily monitoring of GLY and its metabolite AMPA in wheat flour and oats samples.

External Quality Assessment of Glycated Hemoglobin in Iran: Comparison of Five Different Commercial Methods with Two Different Total Allowable Errors.

SummaryBackgroundGlycated hemoglobin (HbA1c) measuring has a critical role in the monitoring and diagnosis of diabetes. So, the analytical performance of its measuring method must be acceptable. Clinical laboratories should continuously monitor the performance of their commercial methods, both by using proper internal quality control (IQC) and by participating in external quality assessment schemes (EQAS).MethodsIn January and August 2016, two different freshly prepared commutable patient QC samples were sent to over 1000 laboratories, but 682 and 925 different laboratories which were used five common commercial methods for measuring HbA1c, included in this study during 23th and 24th runs of the external quality assessment program (EQAP), respectively. Target values for total group and also for peer groups were calculated. The performance of each method and laboratory were determined according to two different allowable total errors (TEa), including ±6% and ±20%, which are suggested by the National Glycohemoglobin Standardization Program (NGSP) and Reference Health Laboratory of Iran, respectively.ResultsConsidering TEa of ±20% in evaluating HbA1c commercial methods and laboratory performances, pass rates ranged from 97% to 98% during EQAP-23 and EQAP-24, respectively. But when this evaluation was performed according to TEa of ±6%, pass rates decreased significantly to 60% and 62%, respectively.ConclusionsUsing improper analytical goals has led to misinterpretation of EQA results. In order to maintain the clinical usefulness of HbA1c results, we need to reduce TEa of ±20% to ±6% and improve HbA1c measuring method performance. Although, with TEa of ±6% our pass rates are not so bad.

Liquid chromatography-tandem mass spectrometry analysis of 17-hydroxyprogesterone in dried blood spots revealed matrix effect on immunoassay.

Immunoassays for measuring 17-hydroxyprogesterone (17-OHP) produce high rates of false positives that impact the identification of congenital adrenal hyperplasia (CAH) in neonates. A confirmatory test with high analytical specificity employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology is needed in newborn screening for CAH. 17-OHP and cortisol were extracted from dried blood spot (DBS) samples, resolved on a C18 column, and measured using tandem mass spectrometry. The results were compared with those determined using the AutoDELFIA immunoassay. The LC-MS/MS method had a limit of quantitation of 10.0 and 5.0ng/mL for 17-OHP and cortisol, respectively. The method characteristics showed coefficient variation (%CV) ≤ 11.9% for both 17-OHP and cortisol, recoveries ranging from 83.1 to 101.5% for 17-OHP and from 95.1 to 102.8% for cortisol, and linearity with R2 = 0.9994 for 17-OHP and R2 = 0.9996 for cortisol, clinical sensitivity of 100.0% and a specificity of 96.4% as obtained by receiver operating characteristic analysis on 45 patient samples when 17-OHP > 39.1ng/mL was selected as the cutoff value. Comparison between the LC-MS/MS and the AutoDELFIA immunoassay methods revealed a poor correlation for patient DBS samples (R2 = 0.6784); however, an excellent correlation was obtained for QC and proficiency test (PT) DBS samples (R2 = 0.9797). The LC-MS/MS method produced reliable results for 17-OHP and cortisol for the diagnosis of CAH. The AutoDELFIA immunoassay appears to be subject to matrix effects in the analysis for 17-OHP in DBS patient samples. The DBS samples of non-patient origin may not be suitable for assessing analytical accuracy of immunoassays.