HPLC and mass spectrometric characterization of a candidate reference material for the alcohol biomarker carbohydrate-deficient transferrin (CDT).

BACKGROUND: Measurement of the alcohol-induced change of the serum transferrin glycoform pattern, carbohydrate-deficient transferrin (CDT), is used as a biomarker for heavy drinking. This study characterized a candidate reference material for CDT measurement derived from isolated human transferrin glycoforms. METHODS: Four transferrin glycoforms were separated from human plasma by standard methods. The identity and purity of the fractions was evaluated by HPLC, using specific absorbance measurement of the iron-transferrin complex at 470 nm, and by mass spectrometry, using ESI Q-Tof MS. A primary candidate reference material was prepared by mixing isolated fractions in transferrin-free plasma in a proportion similar to that in serum and with 0-12% disialotransferrin. A secondary candidate reference material was prepared by spiking a serum pool with 1-9% disialotransferrin. RESULTS: Initial identification of the isolated transferrin fractions as disialo-, trisialo-, tetrasialo- and pentasialotransferrin was based on agreement with established HPLC retention times for authentic serum samples (RRT 0.998-1.004). The presence of single symmetric peaks suggested that the fractions were sufficiently pure. The identity and purity was further based on MS agreement of observed with theoretical molecular masses (Delta(m)<0.03%). The %disialotransferrin target values for the secondary candidate reference material showed good correlation with the measured results by an HPLC candidate reference method (r(2)=0.999). CONCLUSIONS: The separated human transferrin fractions used to prepare the CDT candidate reference material were indicated to contain distinct glycoforms. Having access to a CDT reference material in serum matrix will facilitate comparison of results between different methods and aid in the standardization process.

Adaptation of a high-performance liquid chromatographic method for quantitative determination of homocysteine in urine.

A modification of the Bio-Rad total homocysteine HPLC-test is presented in order to enable not only plasma homocysteine measurements but also the quantification of homocysteine in urine samples using the same principle of measurement. Coelution of the internal standard provided in the test kit with an endogenous compound in urine demands for an alternative analytical procedure. Therefore, we introduced 3-mercaptopropionic acid as a substitute for the internal standard. The analytical method validation was performed for the matrix of urine specimens. The applicability of this method was demonstrated in a clinical study with volunteers after homocysteine thiolactone hydrochloride loading.

Analytical and clinical evaluation of the Bio-Rad HPLC kit for measurement of type I collagen cross links.

The measurement of hydroxylysylpyridinoline (PYD) and lysylpyridinoline (DPD), the degradation products of type I collagen, by manual HPLC assay posed practical difficulties. The present study was undertaken to evaluate the first commercially available HPLC kit, which provides a convenient substitute for cumbersome classical HPLC methods. The HPLC procedure is based on an improved sample clean-up chromatography, convenient ready-to-use HPLC reagents, and quicker isocratic elution of PYR and DPD on reverse-phase analytical column. The analytical parameters assessed include sensitivity, within- and between-assay variation, method comparison, recoveries, and interference. Clinical evaluation included discriminatory power of PYD and DPD and response to treatment of osteoporosis patients with Alendronate. DPD and PYD concentrations showed linear ( r(2) > 0.99) response between 10-400 pmol/ml and 75-4000 pmol/ml, respectively. The average within-assay imprecision, over a range of clinically relevant cross-links concentrations, was CV < 7% ( n = 30). The total imprecision ( n = 35 days), by ANOVA, for PYD and DPD was CV < 7.5% and CV < 10%, respectively. Average spike recovery was 95.4% +/- 6.5%. Comparison with the historical HPLC method exhibited a close correlation ( r values between 0.87 and 0.91, P < 0.0001). Creatinine-corrected DPD in postmenopausal ( Z score = 2.4, P < 0.05, n = 17) and osteoporotic ( Z score = 3.0, P < 0.01, n = 29) women were 44% and 64% higher, respectively, compared to premenopausal samples ( n = 15). Similarly, PYD concentration was 26% and 54% higher in postmenopausal and postmenopausal osteoporotic women, respectively. There was a 47% ( P < 0.001) decrease in DPD concentration ( n = 16), and a 30% decrease in PYD concentration after 90 days of treatment of osteoporotic patients with Alendronate. DPD concentration correlated with N-telopeptide with an r value of 0.69 ( n = 67, P < 0.0001). The reported kit method is substantially simpler and precise than the manual method. DPD concentrations determined by the current method reaffirm the clinical value in identifying increased bone resorption in pathological conditions and monitoring response to antiresorptive therapy.