Report from the HarmoSter study: different LC-MS/MS androstenedione, DHEAS and testosterone methods compare well; however, unifying calibration is a double-edged sword.

OBJECTIVES: Current liquid chromatography-tandem mass spectrometry (LC-MS/MS) applications for circulating androgen measurements are technically diverse. Previously, variable results have been reported for testosterone. Data are scarce for androstenedione and absent for dehydroepiandrosterone sulfate (DHEAS). We assessed the agreement of androstenedione, DHEAS and testosterone LC-MS/MS measurements among nine European centers and explored benefits of calibration system unification. METHODS: Androgens were measured twice by laboratory-specific procedures in 78 patient samples and in EQA materials. Results were obtained by in-house and external calibration. Intra- and inter-laboratory performances were valued. RESULTS: Intra-laboratory CVs ranged between 4.2-13.2 % for androstenedione, 1.6-10.8 % for DHEAS, and 4.3-8.7 % and 2.6-7.1 % for female and male testosterone, respectively. Bias and trueness in EQA materials were within ±20 %. Median inter-laboratory CV with in-house vs. external calibration were 12.0 vs. 9.6 % for androstenedione (p<0.001), 7.2 vs. 4.9 % for DHEAS (p<0.001), 6.4 vs. 7.6 % for female testosterone (p<0.001) and 6.8 and 7.4 % for male testosterone (p=0.111). Median bias vs. all laboratory median with in-house and external calibration were -13.3 to 20.5 % and -4.9 to 18.7 % for androstenedione, -10.9 to 4.8 % and -3.4 to 3.5 % for DHEAS, -2.7 to 6.5 % and -11.3 to 6.6 % for testosterone in females, and -7.0 to 8.5 % and -7.5 to 11.8 % for testosterone in males, respectively. CONCLUSIONS: Methods showed high intra-laboratory precision but variable bias and trueness. Inter-laboratory agreement was remarkably good. Calibration system unification improved agreement in androstenedione and DHEAS, but not in testosterone measurements. Multiple components, such as commutability of calibrators and EQA materials and internal standard choices, likely contribute to inter-laboratory variability.

An LC-MS/MS method for serum cystatin C quantification and its comparison with two commercial immunoassays.

OBJECTIVES: The standardization of cystatin C (CysC) measurement has received increasing attention in recent years due to its importance in estimating glomerular filtration rate (GFR). Mass spectrometry-based assays have the potential to provide an accuracy base for CysC measurement. However, a precise, accurate and sustainable LC-MS/MS method for CysC is still lacking. METHODS: The developed LC-MS/MS method quantified CysC by detecting signature peptide (T3) obtained from tryptic digestion. Stable isotope labeled T3 peptide (SIL-T3) was spiked to control matrix effects and errors caused by liquid handling. The protein denaturation, reduction and alkylation procedures were combined into a single step with incubation time of 1 h, and the digestion lasted for 3.5 h. In the method validation, digestion time-course, imprecision, accuracy, matrix effect, interference, limit of quantification (LOQ), carryover, linearity, and the comparability to two routine immunoassays were evaluated. RESULTS: No significant matrix effect or interference was observed with the CysC measurement. The LOQ was 0.21 mg/L; the within-run and total imprecision were 1.33-2.05 % and 2.18-3.90 % for three serum pools (1.18-5.34 mg/L). The LC-MS/MS method was calibrated by ERM-DA471/IFCC and showed good correlation with two immunoassays traceable to ERM-DA471/IFCC. However, significant bias was observed for immunoassays against the LC-MS/MS method. CONCLUSIONS: The developed LC-MS/MS method is robust and simpler and holds the promise to provide an accuracy base for routine immunoassays, which will promote the standardization of CysC measurement.

Coupling immuno-magnetic capture with LC-MS/MS(MRM) as a sensitive, reliable, and specific assay for SARS-CoV-2 identification from clinical samples.

Recently, numerous diagnostic approaches from different disciplines have been developed for SARS-CoV-2 diagnosis to monitor and control the COVID-19 pandemic. These include MS-based assays, which provide analytical information on viral proteins. However, their sensitivity is limited, estimated to be 5 × 104 PFU/ml in clinical samples. Here, we present a reliable, specific, and rapid method for the identification of SARS-CoV-2 from nasopharyngeal (NP) specimens, which combines virus capture followed by LC-MS/MS(MRM) analysis of unique peptide markers. The capture of SARS-CoV-2 from the challenging matrix, prior to its tryptic digestion, was accomplished by magnetic beads coated with polyclonal IgG-α-SARS-CoV-2 antibodies, enabling sample concentration while significantly reducing background noise interrupting with LC-MS analysis. A sensitive and specific LC-MS/MS(MRM) analysis method was developed for the identification of selected tryptic peptide markers. The combined assay, which resulted in S/N ratio enhancement, achieved an improved sensitivity of more than 10-fold compared with previously described MS methods. The assay was validated in 29 naive NP specimens, 19 samples were spiked with SARS-CoV-2 and 10 were used as negative controls. Finally, the assay was successfully applied to clinical NP samples (n = 26) pre-determined as either positive or negative by RT-qPCR. This work describes for the first time a combined approach for immuno-magnetic viral isolation coupled with MS analysis. This method is highly reliable, specific, and sensitive; thus, it may potentially serve as a complementary assay to RT-qPCR, the gold standard test. This methodology can be applied to other viruses as well.

Eco-Friendly, Simple, Fast, and Sensitive UPLC-MS/MS Method for Determination of Pexidartinib in Plasma and Its Application to Metabolic Stability.

Pexidartinib is the first drug approved by the U.S. Food and Drug Administration specifically to treat the rare joint tumor tenosynovial giant cell tumor. In the current study, a validated, selective, and sensitive UPLC-MS/MS assay was developed for the quantitative determination of pexidartinib in plasma samples using gifitinib as an internal standard (IS). Pexidartinib and IS were extracted by liquid-liquid extraction using methyl tert-butyl ether and separated on an acquity BEH C18 column kept at 40 °C using a mobile phase of 0.1% formic acid in acetonitrile: 0.1% formic acid in de-ionized water (70:30). The flow rate was 0.25 mL/min. Multiple reaction monitoring (MRM) was operated in electrospray (ESI)-positive mode at the ion transition of 418.06 > 165.0 for the analyte and 447.09 > 128.0 for the IS. FDA guidance for bioanalytical method validation was followed in method validation. The linearity of the established UPLC-MS/MS assay ranged from 0.5 to 1000 ng/mL with r > 0.999 with a limit of quantitation of 0.5 ng/mL. Moreover, the metabolic stability of pexidartinib in liver microsomes was estimated.

Bifunctionalized isotopologs as calibrants for standard-free quantitation of drug metabolites in plasma by liquid chromatography coupled with radiometry and mass spectrometry.

The reported method involves a novel workflow that eliminates the need for authentic reference standards for the quantitation of drug metabolites in biological samples using a single multi-isotopically labeled compound bearing both radio and stable isotopes. The resulting radio and stable bifunctionalized isotopolog (RADSTIL) of the parent drug is employed as a substrate for in vitro biotransformation to targeted RADSTILs of metabolites as calibrants. Inclusion of a radio label enables both radiometric and mass spectrometric detection. The addition of stable labels ensures the subsequent isotopic interference-free quantitation of unlabeled metabolites in preclinical and clinical samples. This affords a more accurate quantitation workflow compared with the current semi-quantitation method, which utilizes isotopic interfering radio isotopologs of metabolites alone as calibrants. The proof-of-concept is illustrated with (14 C,13 C2 )-acetaminophen where in vitro biotransformation produced (14 C,13 C2 )-sulfate and (14 C,13 C2 )-glucuronide calibrants. Absolute quantitation of the acetaminophen metabolites was then achieved by liquid chromatography coupled with radiometry and mass spectrometry. Quantitative data obtained by this method fell within 82-86% of the values from conventional LC-MS/MS method.

Evaluation of Differential Peptide Loading on Tandem Mass Tag-Based Proteomic and Phosphoproteomic Data Quality.

Global and phosphoproteome profiling has demonstrated great utility for the analysis of clinical specimens. One barrier to the broad clinical application of proteomic profiling is the large amount of biological material required, particularly for phosphoproteomics─currently on the order of 25 mg wet tissue weight. For hematopoietic cancers such as acute myeloid leukemia (AML), the sample requirement is ≥10 million peripheral blood mononuclear cells (PBMCs). Across large study cohorts, this requirement will exceed what is obtainable for many individual patients/time points. For this reason, we were interested in the impact of differential peptide loading across multiplex channels on proteomic data quality. To achieve this, we tested a range of channel loading amounts (approximately the material obtainable from 5E5, 1E6, 2.5E6, 5E6, and 1E7 AML patient cells) to assess proteome coverage, quantification precision, and peptide/phosphopeptide detection in experiments utilizing isobaric tandem mass tag (TMT) labeling. As expected, fewer missing values were observed in TMT channels with higher peptide loading amounts compared to lower loadings. Moreover, channels with a lower loading have greater quantitative variability than channels with higher loadings. A statistical analysis showed that decreased loading amounts result in an increase in the type I error rate. We then examined the impact of differential loading on the detection of known differences between distinct AML cell lines. Similar patterns of increased data missingness and higher quantitative variability were observed as loading was decreased resulting in fewer statistical differences; however, we found good agreement in features identified as differential, demonstrating the value of this approach.

Simultaneous estimation of ZY-19489 and its active metabolite ZY-20486 in human plasma using LC-MS/MS, a novel antimalarial compound.

Aim: ZY-19489 is a new antimalarial drug candidate and selective LC-MS/MS method was established for estimation of ZY-19489 and its metabolite in human plasma. Materials & methods: LLE was employed for extraction, mass spectrometric quantification performed using positive ionization mode and DCP-IMP was used as an internal standard. The chromatographic separation was achieved using mobile phase 5 mM ammonium formate in water and 0.1% v/v ammonia solution in methanol:acetonitrile (90:10% v/v) and column Agilent Zorbex Extended C18, 3.5 µm, 100 × 4.6 mm with a 6-min run time. Results: The calibration curve of ZY-19489 was linear over range 1-500 ng/ml and 2-200 ng/ml for metabolite. Assay was reproducible, selective and devoid of matrix effect. Conclusion: The validated assay was implemented for clinical sample analysis derived from healthy human subjects and parasitemia-induced subjects.

Practical aspect of dimer adduct formation in small-molecule drug analysis with LC-MS/MS.

Aim: Since the MS/MS based detection of small-molecule drugs with poor or even no ion fragmentation is a challenge in bioanalysis, alternative MS/MS detection strategies were in focus of this study and applied in the field of forensic toxicology. Material & methods: Analyte quantification with liquid chromatography-tandem mass spectrometry of problematic drugs was studied by the application of dimer adduct formation and valproic acid (VPA) was used as a model drug. VPA adduct ions could be identified during infusion experiments and the VPA dimer adduct ion was optimized for the detection. Conclusion: Dimer adduct ion formation can be used as an effective way of VPA quantification in human serum. Further, the parallel detection of dimer adduct ions with other adduct ion types can be stated as advantage in LC-MS/MS analysis of problematic drugs.

Simultaneous determination of duloxetine and 4-hydroxy duloxetine glucuronide in human plasma and back-conversion study.

Aim: To develop an LC-MS/MS method for simultaneous determination of duloxetine and its metabolite, 4-hydroxy duloxetine glucuronide (4HDG) in human plasma and to investigate the potential back-conversion of 4HDG to duloxetine using stability study. Materials & methods: The LC-MS/MS method was validated according to the EMA and USFDA Bioanalytical Method Validation Guidelines and applied to pilot bioequivalence study. Results & conclusion: The method validation results were within the acceptance limits. The stability study and incurred sample reanalysis results ruled out the occurrence of back-conversion. The study highlighted the conduct of back-conversion test and the advantages of LC-MS/MS method in terms of sensitivity, specificity and low consumption of organic solvents.

Using mass spectrometry to overcome the longstanding inaccuracy of a commercially-available clinical testosterone immunoassay.

Accurate measurement of testosterone is important for the diagnosis of gonadal disorders in men, women, and children. Testosterone measurement has limited accuracy at low concentrations by most commercially available immunoassays. We aimed to develop an LC-MS/MS assay to address the inaccuracy of the in-house immunoassay observed over the past decade and to replace it with the new assay. Testosterone in serum/plasma was extracted with commercial supported liquid extraction plates. Method validation was performed following the CLSI C62-A guideline. A total of 126 samples were used for method comparison between the Beckman UniCel DxI immunoassay and LC-MS/MS. Results by immunoassay were 20% lower compared with LC-MS/MS and had minimal correlation (R2 = 0.403) with LC-MS/MS below 100 ng/dL. When comparing specimens from the Accuracy-Based Survey from the College of American Pathologists, the newly developed assay agreed well with the CDC reference measurement procedure. In summary, immunoassay measurement of testosterone can be significantly inaccurate, especially at low concentrations. The newly developed LC-MS/MS assay provides accurate results across the entire measurable range.

Multiplexed single-cell proteomics using SCoPE2.

Many biological systems are composed of diverse single cells. This diversity necessitates functional and molecular single-cell analysis. Single-cell protein analysis has long relied on affinity reagents, but emerging mass-spectrometry methods (either label-free or multiplexed) have enabled quantifying >1,000 proteins per cell while simultaneously increasing the specificity of protein quantification. Here we describe the Single Cell ProtEomics (SCoPE2) protocol, which uses an isobaric carrier to enhance peptide sequence identification. Single cells are isolated by FACS or CellenONE into multiwell plates and lysed by Minimal ProteOmic sample Preparation (mPOP), and their peptides labeled by isobaric mass tags (TMT or TMTpro) for multiplexed analysis. SCoPE2 affords a cost-effective single-cell protein quantification that can be fully automated using widely available equipment and scaled to thousands of single cells. SCoPE2 uses inexpensive reagents and is applicable to any sample that can be processed to a single-cell suspension. The SCoPE2 workflow allows analyzing ~200 single cells per 24 h using only standard commercial equipment. We emphasize experimental steps and benchmarks required for achieving quantitative protein analysis.

Characterization of equine liver microsome-generated metabolites of growth hormone secretagogue small molecule ibutamoren.

RATIONALE: Interest in growth hormone secretagogues has intensified during the past several years based on capable, ever-widening investigational applications of recombinant growth hormone in animals and humans. Ibutamoren is a potent, long-acting, selective and orally active non-peptide growth hormone secretagogue, which has a great potential for abuse as a performance-enhancing agent in sports. METHODS: To support drug metabolism and pharmacokinetic studies of chiral pharmaceuticals, it is necessary to combine the resolving power of high-performance liquid chromatography with the sensitivity of mass spectrometric techniques. This paper describes the metabolic conversion of ibutamoren using equine liver microsomes and metabolite characterization using a QExactive high-resolution mass spectrometer. RESULTS: A total of 32 metabolites for ibutamoren (20 phase I and 12 phase II) were detected. The important findings of the current research are as follows: (1) the growth hormone secretagogue ibutamoren was prone to oxidation, resulting in corresponding hydroxylated metabolites; (2) in ibutamoren, the dissociation of the phenyl ring and 2-amino-2-methylpropanamide side chain was also observed; (3) the glucuronic acid conjugates of mono-, di- and trihydroxylated analogues were detected; and (4) no sulfonic acid conjugated metabolites were observed in this study of ibutamoren. CONCLUSIONS: The reported data help in the speedy detection of the growth hormone secretagogue ibutamoren and reveal its illegal use in competitive sports.

Using a stable isotope-labeled internal standard for liquid chromatography-tandem mass spectrometry quantitation of meloxicam in human plasma.

A sensitive and highly efficient LC-ESI-MS/MS method using a stable isotope-labeled internal standard (SIL IS) to detect meloxicam in human plasma was developed and validated. Sample preparation used only 50 µL human plasma with one-step methanol protein precipitation. A gradient mobile phase system was adopted for chromatographic separation on a Poroshell 120 SB-C18 column (2.1 × 50 mm, 2.7 µm). Positive ion pattern was chosen for quantification under multiple reaction monitoring. Ion pairs were [M + H]+ m/z 352.1 → 115.1 for meloxicam and [M + H]+ m/z 355.1 → 187.1 for meloxicam-d3 (SIL IS). Total run time was 4.0 min. Standard curve was linear over a concentration range from 8.00 to 1600 ng mL-1 . This method was fully validated to evaluate its performance, including specificity, carryover, sensitivity, linearity, accuracy, precision, recovery, matrix effects, stability, dilution reliability and incurred sample reanalysis, which provided a reliable basis for pharmacokinetic studies of meloxicam in 28 healthy Chinese volunteers. After a single-dose oral administration of 7.5 mg meloxicam, the main pharmacokinetic parameters were as follows: Cmax , 814.79 ± 201.37 ng mL-1 ; Tmax , 4.54 ± 1.42 h; AUC0-t , 24,572.04 ± 5766.93 ng·h mL-1 ; AUC0-∞ , 25,810.89 ± 6796.60 ng·h mL-1 and t1/2 , 21.11 ± 5.35 h.

Mycotoxin Biomarkers in Pigs-Current State of Knowledge and Analytics.

Farm animals are frequently exposed to mycotoxins, which have many adverse effects on their health and become a significant food safety issue. Pigs are highly exposed and particularly susceptible to mycotoxins, which can cause many adverse effects. For the above reasons, an appropriate diagnostic tool is needed to monitor pig' exposure to mycotoxins. The most popular tool is feed analysis, which has some disadvantages, e.g., it does not include individual exposure. In recent years, the determination of biomarkers as a method to assess the exposure to mycotoxins by using concentrations of the parent compounds and/or metabolites in biological matrices is becoming more and more popular. This review provides a comprehensive overview of reported in vivo mycotoxin absorption, distribution, metabolism and excretion (ADME) and toxicokinetic studies on pigs. Biomarkers of exposure for aflatoxins, deoxynivalenol, ochratoxin A, fumonisins, T-2 toxin and zearalenone are described to select the most promising compound for analysis of porcine plasma, urine and faeces. Biomarkers occur in biological matrices at trace levels, so a very sensitive technique-tandem mass spectrometry-is commonly used for multiple biomarkers quantification. However, the sample preparation for multi-mycotoxin methods remains a challenge. Therefore, a summary of different biological samples preparation strategies is included in that paper.

Development and Validation of Liquid Chromatography-Tandem Mass Spectrometry Methods for the Quantification of Cefquinome, Ceftiofur, and Desfuroylceftiofuracetamide in Porcine Feces with Emphasis on Analyte Stability.

Cefquinome and ceftiofur are ß-lactam antibiotics used for the treatment of bacterial infections in swine. Although these antimicrobials are administered intramuscularly, the exposure of the gut microbiota to these cephalosporins is not well described. This exposure can contribute to the emergence and spread of antimicrobials in the environment and to the possible spread of antimicrobial resistance genes. To assess the impact of drug administration on the intestinal excretion of these antimicrobials it is essential to measure the amounts of native compound and metabolites in feces. Two (ultra)-high-performance liquid chromatography-tandem mass spectrometry ((U)HPLC-MS/MS) methods were developed and validated, one for the determination of cefquinome and ceftiofur and the other for the determination of ceftiofur residues, measured as desfuroylceftiofuracetamide, in porcine feces. The matrix-based calibration curve was linear from 5 ng g-1 to 1000 ng g-1 for cefquinome (correlation coefficient (r) = 0.9990 ± 0.0007; goodness of fit (gof) = 3.70 ± 1.43) and ceftiofur (r = 0.9979 ± 0.0009; gof = 5.51 ± 1.14) and quadratic from 30 ng g-1 to 2000 ng g-1 for desfuroylceftiofuracetamide (r = 0.9960 ± 0.0020; gof = 7.31 ± 1.76). The within-day and between-day precision and accuracy fell within the specified ranges. Since ß-lactam antibiotics are known to be unstable in feces, additional experiments were conducted to adjust the sampling protocol in order to minimize the impact of the matrix constituents on the stability of the analytes. Immediately after sampling, 500 µL of an 8 µg mL-1 tazobactam solution in water was added to 0.5 g feces, to reduce the degradation in matrix.

Development and certification of a reference material for zearalenone in maize germ oil.

Zearalenone (ZEN), an estrogenic mycotoxin produced by several species of Fusarium fungi, is a common contaminant of cereal-based food worldwide. Due to frequent occurrences associated with high levels of ZEN, maize oil is a particular source of exposure. Although a European maximum level for ZEN in maize oil exists according to Commission Regulation (EC) No. 1126/2007 along with a newly developed international standard method for analysis, certified reference materials (CRM) are still not available. To overcome this lack, the first CRM for the determination of ZEN in contaminated maize germ oil (ERM®-BC715) was developed in the frame of a European Reference Materials (ERM®) project according to the requirements of ISO Guide 35. The whole process of CRM development including preparation, homogeneity and stability studies, and value assignment is presented. The assignment of the certified mass fraction was based upon an in-house study using high-performance liquid chromatography isotope dilution tandem mass spectrometry. Simultaneously, to support the in-house certification study, an interlaboratory comparison study was conducted with 13 expert laboratories using different analytical methods. The certified mass fraction and expanded uncertainty (k = 2) of ERM®-BC715 (362 ± 22) µg kg-1 ZEN are traceable to the SI. This reference material is intended for analytical quality control and contributes to the improvement of consumer protection and food safety.

External quality assessment in the absence of proficiency testing: A split-sample testing program experience.

In the field of laboratory medicine, proficiency testing is a vehicle used to improve the reliability of reported results. When proficiency tests are unavailable for a given analyte, an alternative approach is required to ensure adherence to the International Organization for Standardization (ISO) 15189:2012 standard. In this study, we report the results of a split-sample testing program performed as an alternative to a formal PT. This testing method was based on recommendations provided in the Clinical and Laboratory Standards Institute (CLSI) QMS24 guideline. Two different laboratories measured, in duplicate, the heparan sulfate concentration in five samples using ultra-performance liquid chromatography and tandem mass spectrometry. The data analysis to determine the criterion used for the comparability assessment between the two laboratories was based on Appendix E of the QMS24 guideline. Mean interlaboratory differences fell within the maximum allowable differences calculated from the application of the QMS24 guideline, indicating that the results obtained by the two laboratories were comparable across the concentrations tested. Application of the QMS24 split-sample testing procedure allows laboratories to objectively assess test results, thus providing the evidence needed to face an accreditation audit with confidence. However, due to the limitations of statistical analyses in small samples (participants and/or materials), laboratory specialists should assess whether the maximum allowable differences obtained are suitable for the intended use, and make adjustments if necessary.

Optimized procedures for testing plasma metanephrines in patients on hemodialysis.

Diagnosis of pheochromocytomas and paragangliomas in patients receiving hemodialysis is troublesome. The aim of the study was to establish optimal conditions for blood sampling for mass spectrometric measurements of normetanephrine, metanephrine and 3-methoxytyramine in patients on hemodialysis and specific reference intervals for plasma metanephrines under the most optimal sampling conditions. Blood was sampled before and near the end of dialysis, including different sampling sites in 170 patients on hemodialysis. Plasma normetanephrine concentrations were lower (P < 0.0001) and metanephrine concentrations higher (P < 0.0001) in shunt than in venous blood, with no differences for 3-methoxytyramine. Normetanephrine, metanephrine and 3-methoxytyramine concentrations in shunt and venous blood were lower (P < 0.0001) near the end than before hemodialysis. Upper cut-offs for normetanephrine were 34% lower when the blood was drawn from the shunt and near the end of hemodialysis compared to blood drawn before hemodialysis. This study establishes optimal sampling conditions using blood from the dialysis shunt near the end of hemodialysis with optimal reference intervals for plasma metanephrines for the diagnosis of pheochromocytomas/paragangliomas among patients on hemodialysis.

Common Reference-Based Tandem Mass Tag Multiplexing for the Relative Quantification of Peptides: Design and Application to Degradome Analysis of Diphtheria Toxoid.

Currently, animal tests are being used to confirm the potency and lack of toxicity of toxoid vaccines. In a consistency approach, animal tests could be replaced if production consistency (compared to known good products) can be proven in a panel of in vitro assays. By mimicking the in vivo antigen processing in a simplified in vitro approach, it may be possible to distinguish aberrant products from good products. To demonstrate this, heat-exposed diphtheria toxoid was subjected to partial digestion by cathepsin S (an endoprotease involved in antigen processing), and the peptide formation/degradation kinetics were mapped for various heated toxoids. To overcome the limitations associated with the very large number of samples, we used common reference-based tandem mass tag (TMT) labeling. Instead of using one label per condition with direct comparison between the set of labels, we compared multiple labeled samples to a common reference (a pooled sample containing an aliquot of each condition). In this method, the number of samples is not limited by the number of unique TMT labels. This TMT multiplexing strategy allows for a 15-fold reduction of analysis time while retaining the reliability advantage of TMT labeling over label-free quantification. The formation of the most important peptides could be followed over time and compared among several conditions. The changes in enzymatic degradation kinetics of diphtheria toxoid revealed several suitable candidate peptides for use in a quality control assay that can distinguish structurally aberrant diphtheria toxoid from compliant toxoids.

Targeted Protein Quantification Using Parallel Reaction Monitoring (PRM).

Targeted proteomics represents an efficient method to quantify proteins of interest with high sensitivity and accuracy. Targeted approaches were first established for triple quadrupole instruments, but the emergence of hybrid instruments allowing for high-resolution and accurate-mass measurements of MS/MS fragment ions enabled the development of parallel reaction monitoring (PRM). In PRM analysis, specific peptides are measured as representatives of proteins in complex samples, with the full product ion spectra being acquired, allowing for identification and quantification of the peptides. Ideally, corresponding stable isotope-labeled peptides are spiked into the analyzed samples to account for technical variation and enhance the precision. Here, we describe the development of a PRM assay including the selection of appropriate peptides that fulfill the criteria to serve as unique surrogates of the targeted proteins. We depict the sequential steps of method development and the generation of calibration curves. Furthermore, we present the open-access tool CalibraCurve for the determination of the linear concentration ranges and limits of quantification (LOQ).