An accurate measurement method for serum homocysteine measurement by ID-LC/MS/MS and the application of external quality assessment

BackgroundSerum C-peptide results from various routine methods used in China are highly variable, warranting well-performing methods to serve as an accuracy base to improve the harmonization of C-peptide measurements in China. We developed an accurate isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC–MS/MS) method for serum C-peptide measurement and explored its use in harmonization.MethodsAfter protein precipitation with ZnSO4 solution, C-peptide was extracted from serum samples by anion-exchange solid-phase extraction and quantified by ID-LC–MS/MS in positive ion mode. The precision and analytical recovery of the ID-LC–MS/MS method were assessed. Seventy-six serum samples were analyzed using the ID-LC–MS/MS method and six routine immunoassays. Ordinary linear regression (OLR) and Bland-Altman (BA) analyses were conducted to evaluate the relationship between the ID-LC–MS/MS method and routine immunoassays. Five serum pool samples assigned using the ID-LC–MS/MS method were used to recalibrate the routine assays. OLR and BA analyses were re-conducted after recalibration.ResultsThe within-run, between-run, and total precision for the ID-LC–MS/MS method at four concentrations were 1.0%–2.1%, 0.6%–1.2%, and 1.3%–2.2%, respectively. The analytical recoveries for the ID-LC–MS/MS method at three concentrations were 100.3%–100.7%, 100.4%–101.0%, and 99.6%–100.7%. The developed method and the immunoassays were strongly correlated, with all R2 >0.98. The comparability among the immunoassays was substantially improved after recalibration.ConclusionsThe performance of the ID-LC–MS/MS method was carefully validated, and this method can be used to improve the harmonization of serum C-peptide measurements in China.

To find suitable external quality assessment (EQA) materials for serum C-peptide, we evaluated the commutability of five types of processed materials. Seventy-four individual serum samples and 12 processed samples including three EQA samples currently in use, frozen human serum pools (FHSP), and three other kinds of processed samples were prepared by dissolving WHO International Standard Reagent for C-peptide (WHO ISR 13/146) in three different matrixes: 0.05 % bovine serum albumin, fetal bovine serum and human serum pools. Samples were analyzed using the isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method and six widely used immunoassays. The commutabilities of processed materials were assessed according to the difference in bias approach recommended by the IFCC. And the short- and long-term stability of FHSP samples at different temperatures were also evaluated. Out of the five kinds of processed materials, FHSP samples were commutable on most assays. In contrast, the EQA materials currently in use were only commutable on a few immunoassays. Additionally, processed materials derived from WHO ISR 13/146 were found to be un-commutable on over half of immunoassays. The FHSP samples could be stably stored at 4 and-20 °C for at least 16days, and at-80 °C for at least 1 year, but at room temperature only for 12 h. With clarified commutability and stability information, the human serum pool samples along with the developed ID-LC-MS/MS method could be used in the EQA program to promote the comparability among laboratories for C-peptide measurement in China.

Accurate measurements of plasma mycophenolic acid (MPA) are essential for therapeutic drug monitoring in transplant recipients and autoimmune diseases. The performance of plasma mycophenolic acid routine methods remains highly variable that calls for a candidate reference measurement procedure (cRMP) to improve the standardization of plasma mycophenolic acid measurements. In this study, sample preparation was based on protein precipitation with methanol followed by further dilution. The mycophenolic acid was quantified by the isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) with electrospray ionization in positive ion mode. According to the Clinical and Laboratory Standards Institute (CLSI) documents C62-A and C50-A, the basic analytical performance of the candidate reference method was verified, such as linearity, limit of quantification, matrix effect, precision, accuracy, and uncertainty. Moreover, the candidate reference measurement procedure was compared with the routine liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in a clinical laboratory. Based on the data, the mycophenolic acid in human plasma was well detected by ID-LC-MS/MS. No apparent interferences were found with the mycophenolic acid measurement. The calibration curve for the mycophenolic acid was linear in the concentration range of 0.1-50μg/mL with a correlation coefficient of 0.9999 under the optimum experimental conditions. This method was sensitive because the low limit of quantitation (LOQ) was 0.05μg/mL. The recoveries of MPA were 98.11-98.95%. The intra-day and inter-day coefficients of variations (CV) of our method were ≤ 1.53% and ≤ 0.51%, respectively. No obvious matrix effect was observed. There was a good correlation between this method and the clinical routine LC-MS/MS method. To sum up, we established and validated a reliable plasma MPA method using ID-LC/MS/MS. The desirable accuracy and precision of this method enable it to serve as a promising cRMP to improve the standardization for plasma MPA routine measurements.

Commutability of possible external quality assessment materials for progesterone measurement

Accurate and precise glucose measurements are requisite for ensuring appropriate diagnosis and management of diseases related to hyperglycemia or hypoglycemia. It is necessary to have a higher order method to provide an accuracy base to which routine methods can be compared. We developed and evaluated a highly reliable measurement procedure based on isotope dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS) with a simple one-step derivatization. An appropriate amount of serum was accurately weighed and spiked with an isotope-labeled internal standard. After protein precipitation, the supernatant was reacted with 1-phenyl-3-methyl-5-pyrazolone for chemical structural transformation. The glucose derivatives were analyzed with LC-MS/MS in positive electrospray ionization mode. The within-run and total CVs ranged from 0.28 to 0.42% and from 0.42 to 0.76%, respectively, for a concentration range of 1.691 to 15.676mmol/L. A regression comparison of the presented method to an existing RMP based on ID GC-MS showed agreement with no statistical difference (Y = 0.9985X-0.008; 95 % CI for the slope, 0.9966 to 1.001; 95 % CI for the intercept, -0.012 to 0.019). The structural analogs of glucose with a molecular mass of 180 were tested, and no significant interference effect was found. The limit of quantification was estimated to 0.8ng glucose in absolute amount. This method is accurate, simple, and can serve as a candidate reference measurement procedure (RMP) in the establishment of a serum glucose reference system.

An analytical protocol based on isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS), which includes a peptide-based calibration strategy, was developed and validated for the determination of cardiac troponin I (cTnI) levels in clinical samples. Additionally, thedeveloped method was compared with a protein-based calibration strategy, using cTnI serving as a model for low-abundant proteins. The aim is to evaluate new approaches for protein quantification in complex matrices, supporting the metrology community in implementing new methods and developing fit-for-purpose SI- traceable peptide or protein primary calibrators. To establish traceability to SI units, peptide impurity correction amino acid analysis (PICAA) was conducted to determine the absolute content of signature peptides in the primary standards. Immunoaffinity enrichment was used to capture cTnI from human serum, with a comparison between microbeads and nanobeads to improve enrichment efficiency. Parallel reaction monitoring was used to monitor two signature peptides specific to cTnI. Various digestion parameters were optimized to achieve complete digestion. The analytical method demonstrated selectivity and specificity, allowing the quantification of cTnI within 0.9-22.0 μg/L. The intermediate precision RSD was below 28.9 %, and the repeatability RSD was below 5.8 % at all concentration levels, with recovery rates ranging from 87 % to121 %. The comparison of calibration strategies showed similar LOQ values, but the peptide-based calibration exhibited significant quantitative bias in recovery rates. The data are available via ProteomeXchange (PXD055104). This isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method, based on peptide calibration, successfully quantified cTnI in human serum. Comparing this with protein-based calibration highlighted both the strengths and potential limitations of peptide-based strategies.

Quantification of erufosine, the first intravenously applicable alkylphosphocholine, in human plasma by isotope dilution liquid chromatography–tandem mass spectrometry using a deuterated internal standard

“Systematic review of viscoelastic testing (TEG/ROTEM) in obstetrics and recommendation from the women's SSC of the ISTH”: Response to comment from Kitchen et al

Accurate and precise cortisol measurements are requisite for ensuring appropriate diagnosis and management of diseases related with adrenal or pituitary gland disorders. Prompted by the needs in characterization of certified reference materials and quality assurance for serum cortisol measurements, we developed and evaluated a highly reliable measurement procedure based on isotope dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS) combined with dextran sulfate-Mg2+ precipitation as the sample pretreatment. An appropriate amount of serum was accurately weighed and spiked with the isotope labelled internal standard. After precipitation, massive lipids and lipoproteins were removed from serum matrix. The clear supernatant was transferred and extracted with ethyl acetate-hexane solution. The cortisol was analyzed with LC-MS/MS in positive electrospray ionization mode. The within-run and total coefficient of variations (CVs) ranged from 0.3 to 0.6% and 0.7 to 1.2%, respectively, for a concentration range of 76.30 to 768.04nmol/L. A regression comparison of the results obtained by the present method and the certified values of ERM-DA451 showed agreement with no statistical difference (Y = 1.0092 X-0.7455; 95% CI for the slope, 0.9940 to 1.0212; 95% CI for the intercept, - 3.6575 to 2.6390, r2 = 0.999). All structural analogs of cortisol tested were well resolved from cortisol in 12min on a phenyl ligand column under an isocratic elution. The limit of quantification was estimated to 5pg cortisol in absolute amount. This method is accurate and simple and can be served as a candidate reference measurement procedure in establishment of serum cortisol reference system.

Regorafenib (BAY 73-4506, commercial name Stivarga(®)) is an oral multikinase inhibitor developed by Bayer Pharma AG (Germany) that targets angiogenic, stromal and oncogenic receptor tyrosine kinases. An isotope dilution liquid chromatography-tandem mass spectrometry method has been developed and validated for the simultaneous determination of regorafenib and its two major metabolites BAY 75-7495 (M-2) and BAY 81-8752 (M-5) in lithium-heparinized human plasma. Analysis was performed after protein precipitation on a triple-quadrupole tandem mass spectrometer. The validated concentration range was from 2.00 (lower limit of quantitation) to 2000 µg/l for all the analytes. Selectivity and specificity in the presence of four potential comedications were demonstrated. Interassay accuracy and precision coefficient of variation ranged from 91.2% (at LLOQ of M-5) to 105% and 3.59 to 14.1%, respectively. The method proved to be selective, specific, sufficiently sensitive, highly reproducible and robust for the analysis of large numbers of samples. The method performance met current guidelines on bioanalytical method validation.

Background Treosulfan and busulfan are alkylating agents administered as the conditioning regimen before hematopoietic stem cell transplantation. High busulfan exposure is associated with drug toxicity and low busulfan exposure with rejection or disease recurrence. There is also an association between treosulfan exposure and early toxicity, such as skin toxicity and mucositis. As both busulfan and treosulfan have narrow therapeutic ranges, therapeutic drug monitoring (TDM) is essential. Since the clinical laboratory tests for both drugs are few, a method that can simultaneously measure both drug concentrations under the same conditions would be helpful in practice. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous measurement of treosulfan and busulfan was developed and validated. Methods For preparing calibrators and quality control materials, commercialized reference standards were used for treosulfan, an active metabolite of treosulfan, S,S-EBDM, and busulfan. Treosulfan-D4 and busulfan-D8 isotopes were used as internal standards (ISs). The analytes were separated from 25 µL of plasma after protein precipitation with acetonitrile containing ISs. After centrifugation, the supernatant was diluted with 3% formic acid and then injected into the liquid chromatography system followed by tandem mass spectrometry. Chromatographic separation was performed using ACQUITY Ι-Class plus ultraperformance liquid chromatography (UPLC) system coupled with a XEVO TQ-XS mass spectrometer operated in positive ion electrospray ionization mode. The analytes and ISs were detected in the multiple reaction monitoring (MRM) mode. The limit of detection (LoD), limit of quantification (LoQ), selectivity, linearity, accuracy, precision, carryover, matrix effect, and sample stability were validated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Results Retention times for treosulfan, S,S-EBDM and IS for both analytes were 0.44 min, 0.43 min, and 0.44 min, respectively. Retention times for busulfan and IS for busulfan were 1.20 min and 1.18 min, respectively. The mass-to-charge (m/z) transitions used for quantification in the MRM mode were as follows: treosulfan 296.2 > 87.1; S,S-EBDM 200.2 > 87.1; and busulfan 264.1 > 55.1. The total run time was 4 min. The LoDs of treosulfan, S,S-EBDM, and busulfan were 0.21, 0.23, and 0.001 µg/mL, respectively. The linear range of the calibration curves of treosulfan, S,S-EBDM, and busulfan spanned concentrations of 3.13–100, 0.63–5.00, and 0.16–5.00 µg/mL, respectively. The total precision of the developed method fulfilled the analytical criteria, except for S,S-EBDM. The LC-MS/MS method was adequately selective and accurate and showed no carry-over. It provided an acceptable matrix effect for treosulfan and busulfan; however, the matrix effect could not be excluded for S,S-EBDM. Busulfan and treosulfan were stable in plasma for 2 h at room temperature. When the samples were stored at 10 °C, busulfan was stable for up to a week and treosulfan was stable for up to 3 days, and both were stable for up to a week when stored frozen at −20 °C. Conclusion We developed the LC-MS/MS method to simultaneously measure treosulfan and busulfan. The method met the validation requirements of the CLSI guidelines and showed good performance. Therefore, this method is expected to be helpful in TDM after treosulfan or busulfan treatment in clinical laboratories.

Midostaurin is an oral multitargeted tyrosine kinase inhibitor for the treatment of acute myeloid leukemia (AML). Therapeutic drug monitoring of midostaurin may support its safe use when suspecting toxicity or combined with strong CYP3A4 inhibitors. A stable isotope dilution liquid chromatography-tandem mass spectrometry method was developed and validated for the determination and quantification of midostaurin in human plasma and serum. Midostaurin serum concentrations were analyzed in 12 patients with FMS-like tyrosine kinase 3 (FLT3)-mutated AML during induction chemotherapy with cytarabine, daunorubicin, and midostaurin. Posaconazole was used as prophylaxis of invasive fungal infections. Linear quantification of midostaurin was demonstrated across a concentration range of 0.01-8.00 mg/L. Inter- and intraday imprecisions of the proposed method were well within ±10%. Venous blood samples were taken in nine and three patients in the first and second cycle of induction chemotherapy. Median (range) midostaurin serum concentration was 7.9 mg/L (1.5-26.1 mg/L) as determined in 37 independent serum specimens. In a real-life cohort of AML patients, interindividual variability in midostaurin serum concentrations was high, highlighting issues concerning optimal drug dosing in AML patients. A personalized dosage approach may maximize the safety of midostaurin. Prospective studies and standardization of analytical methods to support such an approach are needed.

Determination of six volatile fatty acids in human serum, urine and faeces by low temperature derivatisation combined with HPLC–MS/MS

Urine albumin is measured in clinical laboratories by immunoturbidimetry. However, large biases are observed among the different routine methods. To standardize the measurement of urine albumin, a reference measurement procedure (RMP) and urine albumin certified reference materials (CRMs) are needed. A candidate RMP for urine albumin based on liquid chromatography-isotope dilution tandem mass spectrometry (LC-IDMS/MS) using human serum albumin as calibration standard was developed. Isotope-labeled human albumin was used as internal standard. Urine samples were digested using trypsin and eight resulting "signature" peptides of albumin were quantified by LC-IDMS/MS. The candidate RMP was employed in value assignment of external quality assessment (EQA) samples and certification of urine albumin reference materials. The commutability of the developed CRMs was assessed against patient samples. The candidate RMP (recovery 101.5-103.2% and CV 1.2-3.3% at about 7-40mg/L) met optimal performance goal. The lower limit of quantification was 0.03mg/L as determined by signal-to-noise method. The EQA results from clinical laboratories using different immunoturbidimetric methods were generally comparable with assigned target values determined by the candidate RMP, with albumin concentrations ranging from 5 to 226mg/L. Urine albumin reference materials (two levels) certified using thecandidate RMP showed good commutability in a preliminary study. With optimal method precision and trueness, as well as comparability with routine methods, the developed RMP may be used for value assignment of EQA samples or certification of reference materials, which are important pillars in urine albumin method standardization.

There is a requirement for accredited laboratories to participate in external quality assessment (EQA) schemes, but there is wide variation in understanding as to what is required by the laboratories...