Internal Quality Control Samples Research Articles

Development, validation and clinical implementation of a UPLC-MS/MS bioanalytical method for simultaneous quantification of cabotegravir and rilpivirine E-isomer in human plasma

A reversed phase ultra-high performance liquid chromatography method was developed for the simultaneous quantification of cabotegravir (CAB) and the E-isomer of rilpivirine (RPV) in human EDTA plasma, also considering RPV E-isomer instability. Because of the instability of RPV (and CAB) in all light conditions, the (RPV Z-isomer/total RPV)-isomer ratio of RPV was determined for each stock, calibration curve standard, quality control sample and patient sample. [2H3]-CAB and [13C6]-RPV were used as internal standard. Sample preparation involved protein precipitation of plasma using methanol. An HSS T3 column with a guard column (set at 40 °C) was used for analyte separation. The mobile phase components were 65 % 0.1 % formic acid in water (A) and 35 % 0.1 % formic acid in acetonitrile (B) and the flow rate was 0.5 mL/min. Detection was performed with tandem mass spectrometry (MS/MS) in a total runtime of 3.0 min. The assay was validated over the concentration range of 0.0500 – 10.0 mg/L for CAB and 0.00300 – 3.00 mg/L for RPV. The average within-day and between-day accuracies for the assay in plasma were 101 % and 101 % for CAB and 97.6 % and 98.5 % voor RPV, respectively. Within-day and between-day precision in coefficients of variations (CV) were 5.0 %. Extraction recovery was 99 % and 102 % for CAB and its internal standard and 95 % and 97 % for RPV and its internal standard. As our aim was that the (Z-isomer RPV/total RPV) response ratio in patient samples had to be less than 10 % to give reliable results, the (Z-isomer RPV/total RPV) response ratio in stocks, calibration curve standards and internal quality control samples were also taken into account being maximal 0.9 % and 2.3 % respectively. This assay has been successfully used in our Therapeutic Drug Monitoring (TDM) service for people living with HIV on long-acting injectable therapy with CAB/RPV and will also be used in future pharmacokinetic studies.

Comparison of Sigma metrics computed by three bias estimation approaches for 33 chemistry and 26 immunoassay analytes.

Sigma metric can be calculated using a simple equation. However, there are multiple sources for the elements in the equation that may produce different Sigma values. This study aimed to investigate the importance of different bias estimation approaches for Sigma metric calculation. Sigma metrics were computed for 33 chemistry and 26 immunoassay analytes on the Roche Cobas 6000 analyzer. Bias was estimated by three approaches: (1) averaging the monthly bias values obtained from the external quality assurance (EQA) studies; (2) calculating the bias values from the regression equation derived from the EQA data; and (3) averaging the monthly bias values from the internal quality control (IQC) events. Sigma metrics were separately calculated for the two levels of the IQC samples using three bias estimation approaches. The resulting Sigma values were classified into five categories considering Westgard Sigma Rules as≥6, <6 and≥5, <5 and≥4, <4 and≥3, and <3. When classifying Sigma metrics estimated by three bias estimation approaches for each assay, 16 chemistry assays at the IQC level 1 and 2 were observed to fall into different Sigma categories under at least one bias estimation approach. Similarly, for 12 immunoassays at the IQC level 1 and 2, Sigma category was different depending on bias estimation approach. Sigma metrics may differ depending on bias estimation approaches. This should be considered when using Six Sigma for assessing analytical performance or scheduling the IQC events.

Determination of sixteen antibiotics and four β-agonists in human urine samples using ultra-performance liquid chromatography-tandem mass spectrometry based on high-throughput automatic solid-phase extraction

An analytical method combining high-throughput automatic solid-phase extraction with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed to determine 16 antibiotics (macrolides, tetracyclines, quinolones, and sulfonamides) and 4 β-agonists (terbutaline, salbutamol, ractopamine, and clenbuterol) in human urine samples. After thawing at room temperature, 1 mL of urine was sampled and the internal standard was added, followed by the addition of 200 μL ammonium acetate buffer and 20 μL β-glucuronidase, and the mixture was incubated at 37 ℃ overnight. Automatic solid-phase extraction was used to extract the target compounds from the urine samples, and the recoveries were compared using different solid-phase extraction 96-well plates (PRiME MCX, Sep-Pak C18, PRiME HLB), types and volumes of rinse solutions and eluents. Satisfactory recoveries of the 20 target compounds were obtained using the Oasis PRiME HLB 96-well plate, with 1.5 mL 10% (v/v) methanol aqueous solution and 2.0 mL methanol as the rinse solution and eluent, respectively. The eluent was concentrated under nitrogen gas at 45 ℃, and the recoveries of the target compounds were compared under different conditions (completely or almost dry, drying to 1 mL, and adding water as a protective agent), and the recovery rate was optimal when water was added as a protective agent. In this study, two types of analytical columns (ACQUITY BEH C18 and ACQUITY HSS T3) and different gradient elution procedures and mobile phases were compared. The optimal chromatographic effect was realized using an HSS T3 column (100 mm×3.0 mm, 1.8 μm) and 0.1% (v/v) formic acid aqueous solution-0.1% (v/v) formic acid in acetonitrile as the mobile phase in gradient elution at a flow rate of 0.3 mL/min. Comparing the peaks observed using different proportions of methanol aqueous solution and the initial mobile phase as the injection solvent revealed that 30% (v/v) methanol aqueous solution was the optimal solution in terms of peak shape and signal-to-noise ratio. MS was conducted using positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode, and the MS parameters were optimized, including the curtain (CUR) and collision gases (CAD). The standard curve obtained using this method exhibited a good linearity (correlation coefficient>0.997), and the respective limits of detection and quantification were 0.02-0.12 ng/mL and 0.06-0.41 ng/mL. At spiked levels of 0.25, 2.5, and 12.5 ng/mL, the recoveries were in the range of 81.7%-120.0% (except that of tetracycline), the intra- and inter-day RSDs (n=6) were 1.1%-11.0% and 1.2%-13.0%, respectively. Azithromycin, trimethoprim, terbutaline, salbutamol, ractopamine, and clenbuterol displayed moderate matrix effects, but all targets exhibited weak matrix effects after correction using the isotope internal standard. To evaluate the accuracy of this method, BCR-503 (containing salbutamol and clenbuterol) and internal quality control samples were used and the concentrations of salbutamol and clenbuterol were within the reference ranges. Additionally, the mean concentrations of the 20 target compounds of two different internal quality control samples after 7 measurements were in the ranges of 0.44-0.59 ng/mL (0.5 ng/mL) and 1.72-2.16 ng/mL (2.0 ng/mL), respectively, which were satisfactory. In this study, the analytical method employed automatic sample pretreatment with a 96-well solid-phase extraction plate, and the detection efficiency was considerably improved. This method displays the advantages of simple operation, ideal recovery, a high sensitivity and weak matrix effect, which satisfies the requirements for the simultaneous determination of 16 antibiotics and 4 β-agonists in human urine samples. This study provides a crucial method for use in monitoring antibiotics and β-agonists in human urine and studying their exposure characteristics and health risks.

Evaluation of Measurement Performance of Routine Chemistry Analytes by Application of Sigma Metrics, Method Evaluation Decision (MEDx) Charts and Quality Goal Index (QGI)

Objective: To evaluate performance of routine chemistry analytes in a tertiary care hospital laboratory by the application of sigma metrics. Introduction: Six sigma (6σ) is a popular Quality Management System (QMS) tool. Laboratories are increasingly using the six sigma method for the objective assessment and comparison of the analytical methods and instrument performance. Six sigma is about measuring or counting the number of defects. It quantifies the performance of a process as a rate of Defects-Per-Million-Opportunities (DPMO or DPM). The aim is to assess the performance and to eliminate or reduce the variation in a process. Materials and Methods: This prospective study was conducted over a period of six months duration. Sigma metrics were calculated using coefficient of variation (%CV), %Bias and total allowable error (%TEa). For %CV we used Internal Quality Control (IQC) samples, at two levels L1 and L2. Daily IQC results of L1 and L2 for 25 routine chemistry analytes were recorded in an excel sheet and %CV was calculated for each analyte for the period of study. For each analyte %Bias was calculated based on values obtained from monthly RIQAS-EQA program data. The total allowable error (%TEa) values for each analyte were extracted from various sources like Clinical Laboratories Improvement Amendment act (CLIA), Canadian Fixed Limits from the College of Physicians and Surgeons of Saskatchewan (CFX) and Spanish Society of Clinical Chemistry and Molecular Pathology (SEQC) table of Desirable Quality Specifications based on Biological Variation (BV) criteria for acceptable performance. Sigma values were calculated. The minimal acceptable performance criteria was considered as 3 sigma. Normalized MEDx charts were used to plot sigma metrics to visually present the performance. Quality Goal Index (QGI) analysis was carried out as a part of Root Cause Analysis (RCA). Results: Highest sigma value of 16.7 was noted for HDL-C and the lowest of 2.08 for chloride at level L1. Many analytes like ALP, Amylase, AST, CK, GGT, HDL-C, Magnesium and Uric Acid attained world class quality performance at both levels L1 and L2 with sigma levels of &gt;6. Many other analytes showed satisfactory performance with sigma levels of &gt;3. Sodium, potassium, chloride and urea did not show a satisfactory performance. Conclusion: Sigma metrics evaluation of analytical performance of our laboratory showed an acceptable performance for a wide range of analytes in patient samples.

Age and gender-specific reference intervals for a panel of lysophosphatidylcholines estimated by tandem mass spectrometry in dried blood spots

Background and objectivesVery long-chain fatty acyl-lysophosphatidylcholines (VLCFA-LysoPCs) are measured in dried blood spots (DBS) for identifying X-linked adrenoleukodystrophy (X-ALD) and other inherited peroxisomal disorders. Our study aimed to establish age- and gender-specific reference intervals for a panel of LysoPCs measured by tandem mass spectrometry in DBS. MethodsLysoPCs (26:0-, 24:0-, 22:0- and 20:0-LysoPCs) were estimated by flow injection analysis-tandem mass spectrometry (FIA-MS/MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods in 3.2 mm blood spots of 2689 anonymized, putative normal subjects (1375 males, and 1314 females) aged between 2 days and 65 years. Samples were divided into groups: Neonates (0–1month), Infants (>1m-1year), Children and Adolescents (>1–18years), and Adults (>18years). Reference intervals were determined using the percentile approach and represented as the median with the 1st and 99th percentile lower and upper limits. ResultsThe percentage coefficient of variation (CV) for repeatability assays of internal and external quality control samples were within acceptable limits. Significant differences (P <0.0001, P <0.01) were observed in the concentrations of 26:0-, 24:0-, 22:0- and 20:0-LysoPCs and their ratios, 26:0/22:0-, 24:0/22:0-, 26:0/20:0-and 24:0/20:0-LysoPC in neonates and infants when compared to children, adolescents, and adults. Levels of 26:0-, 24:0- and 22:0-LysoPCs decreased, whereas 20:0-LysoPC increased with age. There were no significant gender-based differences in the concentration of LysoPCs. ConclusionWe established age- and gender-specific reference intervals for a panel of LysoPCs in DBS. These reference values would be helpful when interpreting LysoPC values in DBS during screening for X-ALD and other peroxisomal disorders.

Efficacy of In-House Quality Control Material Compared to Commercially Available Quality Control for Thyroid Hormone

Laboratory results in clinical laboratories are being generated by automated analyzers. The constant use of commercialcontrol materials is not economically feasible for many countries because of the non-availability or high cost of those materials.Therefore, the preparation of in-house quality control serum will be cost-effective for use in the laboratory. The commercialquality control for immunoassay is costlier and as per NABL 112 criteria, at least two levels of quality control should be run everyday for a laboratory with a large sample load. To overcome this problem our study aims to evaluate the efficacy of in-housepooled serum quality control in comparison with commercial internal quality control samples in thyroid hormone tests. Weprepared in-house quality control from leftover samples of subjects tested for thyroid profile after being screened for HIV, HCV,and HBsAg by pooling them together in a glass jar serum and kept in a deep freezer at -20ºC. Pooled serum was aliquot into 20vials each containing 500 μl. Every day along with commercial internal QC, one aliquot of pooled serum was analyzed for thirtydays for the following parameters: TSH, FT3, FT4. After getting thirty values for each parameter, mean, standard deviation, andcoefficient of variation were calculated for both IQC commercial sample and pooled serum sample.: In-house prepared qualitycontrol material performed as well as commercial quality control for thyroid profile. Therefore, prepared in-house quality controlcan be a good substitute for commercial quality control for thyroid profile.

Verification policies in Croatian medical biochemistry laboratories: a survey of the practice.

IntroductionThe aim of this study was to screen practices used in verification procedures for methods/analysers among medical biochemistry laboratories (MBLs) in Croatia. We hypothesized that these procedures differ widely from laboratory to laboratory and wanted to gather specific data on steps used in the verification workflow.Materials and methodsIn order to obtain data, an online survey was conducted. The survey, divided in two sections, contained 29 questions and statements addressing general characteristics and specific steps of the verification workflow of each individual MBL. The survey was disseminated among managers of all MBLs in Croatia.ResultsA total of 108/196 (55%) laboratories participated in the survey. Forty nine MBLs were excluded from the second part of the survey: 14 have not implemented verification procedures, and 35 MBLs due to the absence of answers. The most relevant results of the second part of the survey showed that: 18/59 (0.31) of the responding MBLs have difficulties when defining acceptance criteria, 27/59 (0.46) used the Clinical and Laboratory Standards Institute protocol for precision estimation; the majority of MBLs used a median of 20 samples for method/analyser comparisons and estimated bias using internal quality control samples; reference intervals provided by external sources are mainly adopted; 60% of MBLs do not include linearity verification in their protocol and do not use the national document for the estimation of measurement uncertainty.ConclusionsHeterogeneous verification protocols are routinely utilized across Croatian MBLs which clearly confirms that a national document might help in the harmonization of verification procedures.

Sensitive Ion-Chromatographic Determination of Citric Acid in Urine

Urine citrate analysis is relevant in the screening and monitoring of patients with calcium nephrolithiasis. A sensitive, fast, easy, and low-maintenance ion chromatographic (IC) method with conductivity detection for the analysis of urine citrate is developed and validated. Its application on true samples is also reported. Sample urine is diluted with a water solution containing internal standard (IS) before the chromatographic assay. The isocratic chromatographic run time is twenty-five minutes, using sodium hydroxide aqueous solution as the mobile phase. The method is fully validated as a quantitative method to objectively demonstrate its applicability for the intended use. The analytical response is linear in the 0.08–10.4 mmol/L concentration range. Precision and accuracy studies carried out on spiked urine and internal quality control samples reveal an imprecision CV% lower than 11% and an accuracy between 85 and 103%. The stability of citrate in urine samples is also evaluated. An easy, rapid, and low-maintenance, cost-effective IC method for urinary citrate determination is developed and validated. Internal standardization improves reliability and precision. The method has been currently used in our laboratory over recent years to analyze more than 1000 samples per year.

Analytical Performance Evaluation of Siemens Sysmex CS-2500 and Sekisui CP-3000.

Automatic coagulation analyzers have been used in the last 50 years and have been developed considerably. Newly developed tests and methods cannot be conducted in routine laboratories without evaluating their performance. Therefore, their performance must be evaluated and approved before being used routinely. The aim of this study is to evaluate the analytical performance of Sysmex Coagulation System-2500 (CS-2500) and Sekisui CP-3000 automatic coagulation analyzer (CP-3000). For APTT, PT, and D-dimer tests, reference range verification study, a method comparison study was performed in both analyzers in accordance with CLSI protocols, and precision and accuracy were evaluated using internal and external quality control samples. In the evaluation of precision and accuracy, CV% and bias% values were calculated. Bland-Altman, Passing-Bablok regression analysis, and correlation coefficient were used in the comparison study. The CV% values calculated for APTT and PT in both analyzers were found to be below the CLSI recommendation of 5%. D-dimer test results meet the quality criteria recommended by CLSI. Accuracy for both analyzers was within the acceptable limits. The reference ranges recommended by the manufacturer have been veryfied. Regression equations for APTT, PT, and D-dimer are y = -3.313 + 1.188x, y = -0.0399 + 1.048x, and y = 0.155 + 0.655x, respectively, and r values were 0.904, 0.978, and 0.974, respectively. CS-2500 and CP-3000 analyzers are suitable for laboratory use for routine coagulation. Since the CP-3000 device is newly used in our country, it needs to be supported by more comparison studies.

Moving average procedures as an additional tool for real-time analytical quality control: challenges and opportunities of implementation in small-volume medical laboratories.

IntroductionMoving average (MA) is one possible way to use patient results for analytical quality control in medical laboratories. The aims of this study were to: (1) implement previously optimized MA procedures for 10 clinical chemistry analytes into the laboratory information system (LIS); (2) monitor their performance as a real-time quality control tool, and (3) define an algorithm for MA alarm management in a small-volume laboratory to suit the specific laboratory.Materials and methodsMoving average alarms were monitored and analysed over a period of 6 months on all patient results (total of 73,059) obtained for 10 clinical chemistry parameters. The optimal MA procedures were selected previously using an already described technique called the bias detection simulation method, considering the ability of bias detection the size of total allowable error as the key parameter for optimization.ResultsDuring 6 months, 17 MA alarms were registered, which is 0.023% of the total number of generated MA values. In 65% of cases, their cause was of pre-analytical origin, in 12% of analytical origin, and in 23% the cause was not found. The highest alarm rate was determined on sodium (0.10%), and the lowest on calcium and chloride.ConclusionsThis paper showed that even in a small-volume laboratory, previously optimized MA procedures could be successfully implemented in the LIS and used for continuous quality control. Review of patient results, re-analysis of samples from the stable period, analysis of internal quality control samples and assessment of the analyser malfunctions and maintenance log have been proposed for the algorithm for managing MA alarms.

Preparation and certification of three new aluminum alloy reference materials using inter-laboratory comparison approach

Accuracy and comparability of measurement results are vital to guarantee correct controlled measurement process and operational conditions. All of these require reliable measurements based on reference materials whose certified values must be gotten by dependable metrological approaches agreeing to the requirements of ISO/ICE 17034 and ISO guide 35 (ISO, 2016; Guide, 2017).The metrological characteristics of certified reference materials (CRMs), especially the uncertainty of their properties, and the position within the traceability hierarchy are items of interest for quality assurance. CRMs play a key role in the quality control, precision studies, verification and validation of test methods. Comparability and the accuracy of the measurement results depend strongly on the availability of certified reference materials appropriate for a specific application. Within the present work, different aluminum alloys reference materials were produced by the National Institute of Standards (NIS), Egypt. Homogeneity of the produced reference materials (RMs) was studied by spark emission spectrometer (AES) and therefore, the results revealed that the produced RMs were homogeneous to disseminate the traceability through the calibration process. Also, they are useful to be used as internal quality control samples to evolve that different products meet required specification. The characterization process of the produced reference materials composition was performed by different independent analytical methods; XRF, Atomic emission spectrometer, inductively coupled plasma- optical emission spectrometer and atomic absorption spectrometry in several laboratories. The statistical data processing, and the data combining in the characterization step is the most important step of reference materials production process. Several methods are used to assign the certified value and its uncertainty such as the robust mean, the weighted mean approaches. The use of inter-laboratory comparison results for reference material certification is very common. In the present study, the assigned values of the mass fractions of the varied elements of the alloys and their uncertainty were calculated by applying the weighted mean approach to inter-laboratory comparison results collected.

Implementation of internal quality control program for monitoring of enzyme-linked immunosorbent assay performance at a blood center

BACKGROUND:Internal quality control (IQC) samples may be incorporated in enzyme-linked immunosorbent assay (ELISA) routinely for detection of errors occurring due to change in environmental conditions, test system, or operator performance. We have described methodology for preparation of IQC samples, monitoring of results using Levey–Jennings (LJ) charts and their interpretation. We have also described our experience of quality control in ELISA using IQC samples, identification of errors and corrections applied.MATERIALS AND METHODS:IQC samples for anti-HIV, hepatitis B surface antigen (HBsAg), and anti-HCV ELISA were prepared “in-house” using standard methodology. After validation of run, E-ratio of IQC sample was calculated and plotted on LJ chart. Further interpretation was done to detect the errors. LJ charts illustrating the performance of IQC samples on 180 runs for each ELISA were drawn and analyzed.RESULTS:For anti-HIV ELISA, violation of warning rule was found in 2 runs (1.11%). Only one run (0.55%) was rejected due to violation of rejection rule. For HBsAg ELISA, violation of warning rule was indicated in two runs (1.11%). Two runs (1.11%) were rejected due to violation of rejection rules. For anti-HCV ELISA, violation of warning rule was indicated in two runs (1.11%), whereas two runs were rejected due to violation of rejection rules. Comprehensive checks were performed for the evaluation of equipment calibration, handling, and storage temperature of reagents and operator's technique. A thorough investigation was undertaken according to the type of error.CONCLUSION:Inclusion of IQC with each ELISA run is valuable to check the assay performance, ensuring reliability and reproducibility of test results.

Quantification of direct-acting oral anticoagulants: Application of a clinically validated liquid chromatography-tandem mass spectrometry method to forensic cases.

In certain forensic cases, a quantification of direct-acting oral anticoagulants (DOACs) can be necessary. We evaluate the applicability of a previously described liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology for the determination of DOACs in plasma to postmortem specimen. Postmortem internal quality control (PIQC) samples were prepared in pooled blank postmortem heart blood, femoral blood, cerebrospinal fluid (CSF), and urine as well in plasma. To examine the application of the clinical method to forensic cases, the main validation parameters were reinvestigated using PIQC samples. Postmortem samples of 12 forensic cases with evidence of previous rivaroxaban intake and unknown bleeding disorders were analyzed. Interday variability remained within the acceptance criterion of ±15%. Matrix effects were comparable in blank plasma and postmortem matrix extracts. After 4 weeks of storage in the refrigerator, no relevant decrease of DOACs was evident. After 96 h of storage at room temperature, a slight decrease in edoxaban concentration was observed in CSF and urine, while plasma edoxaban decreased by about 50%. Median (range) rivaroxaban concentrations determined in specimen of forensic cases were as follows: heart blood (n = 6), 17.2 ng/ml (<LOQ, 56.6 ng/ml); femoral blood (n = 12), 27.6 ng/ml (<LOQ, 110.5 ng/ml); CSF (n = 7), 11.7 ng/ml (<LOQ, 17.5 ng/ml); urine (n = 6), 275.7 ng/ml (14.5-870.9 ng/ml). The median heart/femoral blood rivaroxaban ratio was 1.2 (n = 5). Exemplary, a forensic case with detection of edoxaban in femoral blood, CSF, and urine, is presented. DOACs can be detected in postmortem heart and femoral blood, CSF, and urine specimen by LC-MS/MS. Based on limited forensic cases, no significant redistribution was evident for rivaroxaban, which was found at highest concentrations in urine.

Simultaneous determination of 24,25- and 25,26-dihydroxyvitamin D3 in serum samples with liquid-chromatography mass spectrometry – A useful tool for the assessment of vitamin D metabolism

Vitamin D status is typically assessed by the measurement of 25-hydroxyvitamin D (25(OH)D). However, in selected patient groups the sole determination of 25(OH)D has been proven insufficient for this purpose. The simultaneous measurement of additional vitamin D metabolites may provide useful information for a better evaluation of the vitamin D status. Therefore, we developed and validated a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of 25(OH)D3, 25(OH)D2, 24,25(OH)2D3 and additionally 25,26(OH)2D3, which was identified with a synthesized pure substance.Pure and deuterated substances were used to prepare calibrators and internal standards for all target metabolites. Pre-analytical sample preparation comprised protein precipitation followed by liquid-liquid-extraction and derivatization with 4-Phenyl-1,2,4-triazole-3,5-dione (PTAD) using 50 µL sample volume. Samples were analyzed on an Agilent HPLC 1260 system equipped with a silica-based Kinetex® 5 µm F5 100 Å core-shell column (150 × 4.6 mm) coupled to a Sciex 4500 mass spectrometer.For all four metabolites, limit of detection (LoD) and limit of quantification (LoQ) ranged from 0.3 to 1.5 nmol/L and 1.0 to 3.1 nmol/L, respectively. Recovery varied between 76.1 % and 84.3 %. Intra- and inter-assay imprecision were <8.6 % and <11.5 %, respectively. The analysis of external and internal quality control samples showed good accuracy for 25(OH)D3, 25(OH)D2, 24(R),25(OH)2D3 and 25,26(OH)2D3. Method comparison studies with human samples that were also analyzed with two other LC-MS/MS methods showed close agreement. Finally, the present method has been shown capable of identifying patients with 24-hydroxylase deficiency, which proves its clinical utility.

Internal quality control samples for hair testing

For quality assurance, soaked quality hair samples can serve as good substitutes for the detection of chronic drug use and single-dose intake in the hair testing of multi-analytes when authentic hair samples containing the relevant substances are not available as reference material. In this study, we investigated the soaking technique for 29 common pharmaceuticals and drugs of abuse by exposing drug-free hair to reference standard solutions for a few hours. The incorporated amount had a range of 0.27–4.5 ng/mg (unwashed) following 1 h of exposure in an aqueous standard solution. Following the general washing procedures, 27 %–70 % of the incorporated amount remained in the hair, indicating that the drugs penetrated into the hair cells to some extent and were not only deposited on the surface of the hair. Thus, swelling agents such as water and methanol could allow drugs to diffuse from the solution into the hair cells, similar to the incorporation of drugs from the sebum and sweat coating the growing hair. Following the routine analysis of soaked quality samples at two levels for a one-year period, the samples were proved to be homogenous within a batch, with method imprecision less than 20 % (mean: 12 %) at a low level and less than 17 % (mean: 11 %) at a high level. Furthermore, the monitoring of aliquots of the soaked quality samples in control charts showed that the soaked samples were stable within a year except for the unstable analytes chlordiazepoxide and zopiclone, in which a decline of up to 20 % was observed.

A review of strategies for untargeted urinary metabolomic analysis using gas chromatography-mass spectrometry.

Human urine gives evidence of the metabolism in the body and contains different metabolites at various concentrations. A number of analytical techniques including mass spectrometry (MS) and nuclear magnetic resonance (NMR) have been used to obtain metabolites levels in urine samples. However, gas chromatography-mass spectrometry (GC-MS) is one of the most widely used techniques for urinary metabolomics studies due to its higher sensitivity, resolution, reproducibility, reliability, relatively low cost and ease of operation compared to liquid chromatography-mass spectrometry and NMR. This review looks at various aspects of urine preparation prior to analysis by GC-MS including sample storage, urease pretreatment, derivatization, use of internal standard and quality control samples for data correction. In addition, most common types of inlet liners, ionization techniques and columns are discussed and a summary of mass analyzers are also highlighted. Lastly, the role of retention index in metabolite identification and data normalization methods are presented. The purpose of this review is summarizing methods of sample storage, pretreatment, and GC-MS analysis that are mostly used in urine metabolomics studies. Specific emphasis is given to the critical steps within the GC-MS urine metabolomics that those new to this field need to be aware of and the remaining challenges that require further attention and studies.

Optimization and validation of patient-based real-time quality control procedure using moving average and average of normals with multi-rules for TT3, TT4, FT3, FT3, and TSH on three analyzers.

BackgroundWe have designed a patient‐based real‐time quality control (PBRTQC) procedure to detect analytical shifts and review analytical trends of measurement procedures.MethodsAll the nine months' patient results of total thyroxine (TT4), total triiodothyronine (TT3), free thyroxine (FT4), free triiodothyronine (FT3), and thyrotropin (TSH) measured by three identical analyzers were divided into three groups according to the source of inpatient patients, outpatient patients, and healthy people. The data in each group were truncated by optimized Box‐Plot method and normalized by Box‐Cox method if necessary. The z‐score charts of internal quality control (IQC) samples' results and PBRTQC data were drawn by IQC levels and groups, respectively. The analytical shifts and analytical trends were detected by multi‐rules of 2‐2S rules and moving average rules. The performances of PBRTQC were compared with the BIQC in which IQC samples were measurand only once per day at the beginning of the analytical batch. Twelve quality control cases were listed to validate the performances.ResultsAll the five analytes presented normal distributions when the parameter n of Box‐Plot method was 1.2. The percentages of excluded data ranged from 2.9% to 11.6%. 31 and 14 rejections triggered in PBRTQC and BIQC, respectively. 96.8% of the shift rejections in PBRTQC were trend‐related shifts and calibration‐related shifts, while the proportion was 85.7% in BIQC but 78.6% of the shift rejections in TSH. 25.7% and 8.6% of 105 calibration events which caused analytical shifts were detected by PBRTQC and BIQC, respectively. However, the performance of PBRTQC was not well in TSH because of its large coefficient of variation.ConclusionsThe optimized PBRTQC is high efficiency than BIQC in detecting analytical shifts, trends, and calibration events. The PBRTQC can be used as a low‐cost supplementary procedure to IQC every day, especially at the end of the analytical batch on that day when the within‐individual biological variation of analyte is not larger than its coefficient of variation in IQC. Further optimization and validation of PBRTQC are still needed.

Implementation of quality controls is essential to prevent batch effects in breathomics data and allow for cross-study comparisons

Exhaled breath analysis has become a promising monitoring tool for various ailments by identifying volatile organic compounds (VOCs) as indicative biomarkers excreted in the human body. Throughout the process of sampling, measuring, and data processing, non-biological variations are introduced in the data leading to batch effects. Algorithmic approaches have been developed to cope with within-study batch effects. Batch differences, however, may occur among different studies too, and up-to-date, ways to correct for cross-study batch effects are lacking; ultimately, cross-study comparisons to verify the uniqueness of found VOC profiles for a specific disease may be challenging. This study applies within-study batch-effect-correction approaches to correct for cross-study batch effects; suggestions are made that may help prevent the introduction of cross-study variations. Three batch-effect-correction algorithms were investigated: zero-centering, combat, and the analysis of covariance framework. The breath samples were collected from inflammatory bowel disease (), chronic liver disease (), and irritable bowel syndrome () patients at different periods, and they were analysed via gas chromatography-mass spectrometry. Multivariate statistics were used to visualise and verify the results. The visualisation of the data before any batch-effect-correction technique was applied showed a clear distinction due to probable batch effects among the datasets of the three cohorts. The visualisation of the three datasets after implementing all three correction techniques showed that the batch effects were still present in the data. Predictions made using partial least squares discriminant analysis and random forest confirmed this observation. The within-study batch-effect-correction approaches fail to correct for cross-study batch effects present in the data. The present study proposes a framework for systematically standardising future breathomics data by using internal standards or quality control samples at regular analysis intervals. Further knowledge regarding the nature of the unsolicited variations among cross-study batches must be obtained to move the field further.

Targeted realignment of LC-MS profiles by neighbor-wise compound-specific graphical time warping with misalignment detection.

Liquid chromatography-mass spectrometry (LC-MS) is a standard method for proteomics and metabolomics analysis of biological samples. Unfortunately, it suffers from various changes in the retention times (RT) of the same compound in different samples, and these must be subsequently corrected (aligned) during data processing. Classic alignment methods such as in the popular XCMS package often assume a single time-warping function for each sample. Thus, the potentially varying RT drift for compounds with different masses in a sample is neglected in these methods. Moreover, the systematic change in RT drift across run order is often not considered by alignment algorithms. Therefore, these methods cannot effectively correct all misalignments. For a large-scale experiment involving many samples, the existence of misalignment becomes inevitable and concerning. Here, we describe an integrated reference-free profile alignment method, neighbor-wise compound-specific Graphical Time Warping (ncGTW), that can detect misaligned features and align profiles by leveraging expected RT drift structures and compound-specific warping functions. Specifically, ncGTW uses individualized warping functions for different compounds and assigns constraint edges on warping functions of neighboring samples. Validated with both realistic synthetic data and internal quality control samples, ncGTW applied to two large-scale metabolomics LC-MS datasets identifies many misaligned features and successfully realigns them. These features would otherwise be discarded or uncorrected using existing methods. The ncGTW software tool is developed currently as a plug-in to detect and realign misaligned features present in standard XCMS output. An R package of ncGTW is freely available at Bioconductor and https://github.com/ChiungTingWu/ncGTW. A detailed user's manual and a vignette are provided within the package. Supplementary data are available at Bioinformatics online.

Verification of Quantitative Measurement of Urinary Creatinine Enzymatic Method Kit by DIRUI CS-T240 Clinical Chemistry Analyser

Introduction: All urine samples sent for the diagnosis of inborn errors of metabolism (IEM) disorders testing will be first analysed for their creatinine levels. The urine creatinine analysis is applied either for calculation of urine amount needed in other tests or for quantitation of analytes per mol creatinine. Jaffe kinetic-end point method has been the preferred method. We aim to verify the performance of DIRUI enzymatic method reagent kit on DIRUI CS-T240 analyser and compare with Randox Jaffe kinetic-end point method reagent kit on same platform.Method: Precision study, linearity and method comparisons were carried out using three levels of internal quality control (IQC) samples and the measured results were compared against manufacturer’s claims, medical decision limit and biological variation (BV).Results: The within run coefficient variation (CV) were 3.57%, 2.61% and 1.35% for IQC Level 1, 2 and 3 respectively. The total precision and accuracy were 9.59% and 0.27% for Level 1, 3.17% and 0.57% for Level 2, while 2.03% and 3.63% for Level 3. Whereas, the verifica t ion value for CV based on analytical performance specifications (APS) BV desirable for CV and accuracy were 18.2% and 12.2% respectively. These values were compared, and the precision study was acceptable within desirable specification. Linearity met the manufacturer’s claim and range from 0.32 to 21.2mmol/L. The method comparison gave good correlation, r2 of 0.9975 and regression equation, y=0.9852x-0.022 with medical decision limit, 0.53mmol/L. The assessment of acceptability of this new method was acceptable as calculated total error (TEc) &lt; total allowable error (TEa) (APS BV Desirable for TEa = 42.1%).Conclusion: The analytical verification study that we had carried out fulfilled the BV desirable in precision, accuracy and TEa. Therefore, the performances of enzymatic method kit on DIRUI CS-T240 analyser is verified to be fit-for-purpose and can replace the existing method.International Journal of Human and Health Sciences Supplementary Issue: 2019 Page: 53