Simple and accurate candidate reference measurement procedure for total testosterone in human serum by one-step liquid-liquid extraction coupled with isotope dilution mass spectrometry.

Testosterone in human serum is commonly tested in clinical laboratories using immunoassay methods as well as liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. To standardize and ensure the accuracy of the measurement results, reference procedures with higher metrological order are required. A simple measurement procedure based on one-step liquid-liquid extraction (LLE) and liquid chromatography-isotope dilution tandem mass spectrometry (LC-IDMS/MS) was developed for total testosterone in human serum. The procedure involved serum spiked with 13C3-testosterone, equilibration for 2 h, and extraction with an organic solvent. Testosterone certified reference material (CRM) was used as the calibration standard to ensure the traceability to the International System of Units (SI). Testosterone in serum CRMs from the National Institute for Standards and Technology (NIST) and LGC were used to validate the accuracy of the newly developed method. The deviations of the obtained values from the NIST and LGC certified values ranged from -0.55% to 0.45%. Similarly, the coefficient of variations (CVs) of the replicate measurements were in the range of 0.55% and 0.78%, respectively. The relative expanded uncertainties were comparable with those of the certified materials. The newly developed LC-IDMS/MS procedure demonstrated adequate trueness and precision, and was simple to perform. The method can be used for value assignment of testosterone in external quality assessment (EQA) materials as well as certification of CRMs in the future. Graphical abstract.

Simple and accurate measurement of carbamazepine in surface water by use of porous membrane-protected micro-solid-phase extraction coupled with isotope dilution mass spectrometry.

To achieve fast and accurate analysis of carbamazepine in surface water, we developed a novel porous membrane-protected micro-solid-phase extraction (µ-SPE) method, followed by liquid chromatography-isotope dilution tandem mass spectrometry (LC-IDMS/MS) analysis. The µ-SPE device (∼0.8 × 1 cm) was fabricated by heat-sealing edges of a polypropylene membrane sheet to devise a bag enclosing the sorbent. The analytes (both carbamazepine and isotope-labelled carbamazepine) were first extracted by µ-SPE device in the sample (10 mL) via agitation, then desorbed in an organic solvent (1 mL) via ultrasonication. Several parameters such as organic solvent for pre-conditioning of µ-SPE device, amount of sorbent, adsorption time, and desorption solvent and time were investigated to optimize the µ-SPE efficiency. The optimized method has limits of detection and quantitation estimated to be 0.5 ng L(-1) and 1.6 ng L(-1), respectively. Surface water samples spiked with different amounts of carbamazepine (close to 20, 500, and 1600 ng L(-1), respectively) were analysed for the validation of method precision and accuracy. Good precision was obtained as demonstrated by relative standard deviations of 0.7% for the samples with concentrations of 500 and 1600 ng kg(-1), and 5.8% for the sample with concentration of 20 ng kg(-1). Good accuracy was also demonstrated by the relative recoveries in the range of 96.7%-103.5% for all samples with uncertainties of 1.1%-5.4%. Owing to the same chemical properties of carbamazepine and isotope-labelled carbamazepine, the isotope ratio in the µ-SPE procedure was accurately controlled. The use of µ-SPE coupled with IDMS analysis significantly facilitated the fast and accurate measurement of carbamazepine in surface water.

Developing a reference measurement procedure for free glycerol in human serum by two-step gas chromatography-isotope dilution mass spectrometry.

BACKGROUND: Free glycerol in human serum is measured in clinical laboratories using enzymatic methods, which can be affected by interferences from biological samples. These methods are not applicable when stable isotopic tracers are used to determine lipid kinetics. Hence, a reference measurement procedure for free glycerol in human serum is needed. METHODS: A reference measurement procedure based on two-step gas chromatography-isotope dilution mass spectrometry (GC-IDMS) was developed for the measurement of free glycerol in human serum. This procedure involved spiking with (13)C3-glycerol, protein precipitation and cation exchange SPE, followed by two-step derivatization with 1-butylboronic acid and N-methyl-N-trimethylsilyltrifluoroacetamide. Tripalmitin certified reference material (CRM) was used as the calibration standard to ensure metrological traceability. RESULTS: Good precision and accuracy were obtained as demonstrated by relative standard deviation (RSD) of 1.51%-3.33%, with average recoveries over 98%. The relative measurement uncertainty was below 3% with major contributions from the concentration of glycerol calibration solution, choice of ion pair, linear regression, and measurement precision. CONCLUSIONS: With good accuracy and precision, as well as clear metrological traceability, the developed GC-IDMS procedure is useful in producing traceable and accurate measurement of free glycerol in human serum.

An improved reference measurement procedure for triglycerides and total glycerides in human serum by isotope dilution gas chromatography-mass spectrometry.

BACKGROUND: Triglycerides are widely tested in clinical laboratories using enzymatic methods for lipid profiling. As enzymatic methods can be affected by interferences from biological samples, this together with the non-specific nature of triglycerides measurement makes it necessary to verify the accuracy of the test results with a reference measurement procedure. Several such measurement procedures had been published. These procedures generally involved lengthy and laborious sample preparation steps. In this paper, an improved reference measurement procedure for triglycerides and total glycerides was reported which simplifies the sample preparation steps and greatly shortens the time taken. METHODS: The procedure was based on isotope dilution gas chromatography-mass spectrometry (IDGC-MS)with tripalmitin as the calibration standard. Serum samples were first spiked with isotope-labeled tripalmitin. For the measurement of triglycerides, the serum samples were subjected to lipid extraction followed by separation of triglycerides from diglycerides and monoglycerides. Triglycerides were then hydrolyzed to glycerol, derivatized and injected into the GC­MS for quantification. For the measurement of total glycerides, the serum samples were hydrolyzed directly and derivatized before injection into the GC-MS for quantification. RESULTS: All measurement results showed good precision with CV <1%. A certified reference material (CRM) of lipids in frozen human serum was used to verify the accuracy of the measurement. The obtained values for both triglycerides and total glycerides were well within the certified ranges of the CRM, with deviation <0.4% from the certified values. The relative expanded uncertainties were also comparable with the uncertainties associated with the certified values of the CRM. The validated procedure was used in an External Quality Assessment (EQA) Program organized by our laboratory to establish the assigned values for triglycerides and total glycerides.