Gas chromatography-mass spectrometry (GC-MS) methods have been used extensively in clinical steroid analyses. Evaluating the metabolic ratios of precursors to products by accurate quantification of individual steroid levels in biologicalsamples can reveal the activities of enzymes associated with steroid metabolism. This review article discusses the impact of GCMS-based steroid profiling on our understanding of the biochemical role of steroids and their metabolic enzymes in hormonedependentdiseases, such as congenital adrenal hyperplasia (CAH), cortisol-mediated hypertension, apparent mineralocorticoidexcess (AME), male-pattern baldness, and breast and thyroid cancers. Steroid profiling is a comprehensive analytical techniquethat can be applied whenever the highest specificity is required and may be a reasonable initial diagnostic approach.
A specific and sensitive liquid chromatography-electrospray ionization tandem mass spectrometry method (LC-ESIMS/MS) was developed and validated for the simultaneous quantification of porphyrins (coproporphyrin, pentacarboxylporphyrin,hexacarboxylporphyrin, heptacarboxylporphyrin, and uroporphyrin) in human plasma and urine. Acidified plasma samplesand urine samples were prepared by using liquid-liquid extraction using ethyl acetate and protein precipitation with acetonitrile,respectively. The separation was achieved onto a Synergi Fusion RP column (150 mm × 2.0 mm, 4 μm) with a gradient elutionof mobile phase A (0.1% formic acid in 2 mmol/L ammonium acetate, v/v) and mobile phase B (20% methanol in acetonitrile, v/v) at a flow rate of 450 μL/min. Porphyrins and the internal standard (IS), coproporphyrin I-15N4, were detected by a tandemmass spectrometer equipped with an electrospray ion source operating in positive ion mode. Multiple reaction monitoring(MRM) transitions of the protonated precursor ions and the related product ions were optimized to increase selectivity and sensitivity. The proposed method was validated by assessing selectivity, linearity, limit of quantification (LOQ), precision, accuracy,recovery, and stability. The calibration curves were obtained in the range of 0.1-100 nmol/L and the LOQs were estimated as0.1 nmol/L for all porphyrins. Results obtained from the validation study of porphyrins showed good accuracy, precision, recovery,and stability. Finally, the proposed method was successfully applied to clinical studies on the autism spectrum disorder(ASD) diagnosis of 203 Korean children.
The effectiveness of tertiary matrices composed of the combination of three common matrices (dihydrobenzoic acid(DHB), α-cyano-4-hydroxycinnamic acid (CHCA), and sinapinic acid (SA)) was compared with that of single or binary matricesin the analysis of polyethylene glycol (PEG) polymers ranging from 1400 to 10000 Da using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A tertiary matrix of 2,5-DHB+CHCA+SA was the mosteffective in terms of S/N ratios. CHCA and CHCA+SA produced the highest S/N ratios among the single matrices and thebinary matrices, respectively. The improvement observed when using a tertiary matrix in analyses of PEG polymers by MALDITOFMS is believed to be due to the uniform morphology of the MALDI sample spots and synergistic effects arising from themixture of the three matrix materials.
Glycated hemoglobin (HbA1c) is used as an index of mean glycemia over prolonged periods. This study describes anoptimization of enzyme digestion conditions for quantification of non-glycated hemoglobin (HbA0) and HbA1c as diagnosticmarkers of diabetes mellitus. Both HbA0 and HbA1c were quantitatively determined followed by enzyme digestion using isotopedilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) with synthesized N-terminal hexapeptides asstandards and synthesized isotope labeled hexapeptides as internal standards. Prior to quantification, each peptide was additionallyquantified by amino acid composition analysis using ID-LC-MS/MS via acid hydrolysis. Each parameter was consideredstrictly as a means to improve digestion efficiency and repeatability. Digestion of hemoglobin was optimized when using100 mM ammonium acetate (pH 4.2) and a Glu-C-to-HbA1c ratio of 1:50 at 37oC for 20 h. Quantification was satisfactorilyreproducible with a 2.6% relative standard deviation. These conditions were recommended for a primary reference method ofHbA1c quantification and for the certification of HbA1c reference material.
A gravimetric standard addition method combined with internal standard calibration has been successfully developed forthe accurate analysis of total arsenic in a laver candidate reference material. A model equation for the gravimetric standard additionapproach using an internal standard was derived to determine arsenic content in samples. Handlings of samples, As standard andinternal standard were carried out gravimetrically to avoid larger uncertainty and variability involved in the volumetric preparation. Germanium was selected as the internal standard because of its close mass to the arsenic to minimize mass-dependent bias in massspectrometer. The ion signal ratios of 75As+ to 72Ge+ (or 73Ge+) were measured in high resolution mode (R≥10,000) to separatepotential isobaric interferences by high resolution ICP/MS. For method validation, the developed method was applied to the analysisof arsenic content in the NMIJ 7402-a codfish certified reference material (CRM) and the result was 37.07 mg·kg-1 ± 0.45 mg·kg-1 which is in good agreement with the certified value, 36.7 mg·kg-1 ± 1.8 mg·kg-1. Finally, the certified value of thetotal arsenic in the candidate laver CRM was determined to be 47.15 mg·kg-1 ± 1.32 mg·kg-1 (k = 2.8 for 95% confidence level)which is an excellent result for arsenic measurement with only 2.8 % of relative expanded uncertainty.