Certification and stability assessment of recombinant human growth hormone as a certified reference material for protein quantification.

A certified reference material (CRM) for the quantification of protein, essential to manage quality control and quality assurance in protein-related works, has been developed. Amino acid analysis with conventional acid hydrolysis and isotope dilution HPLC-MS was used to establish an SI-traceable absolute protein quantification method using recombinant human growth hormone (hGH) as a model protein. The certification method was verified by comparative studies between 1) different methods of protein quantification based on microwave-assisted hydrolysis, and 2) different labs as part of the Asian Collaboration on Reference Material project with Japan, China, and Korea. Certification, evaluation of measurement uncertainty, homogeneity testing, and stability testing were carried out, after which the candidate CRM for hGH quantification was successfully certified with excellent agreement within the certified value in the two comparative studies. Although the quantification value of hGH by amino acid analysis showed good robustness in various conditions, results of intact protein analysis showed degradation profiles in temperatures higher than 4 °C. Consequently, storage and dissemination conditions should be set in accordance with stability tests. Based on the results, this method is believed to be suitable for accurate quantification of hGH. Additionally, it can also be used as a guide to preparation of CRM, and instructions for quality management of protein work for other similar proteins.

Measurement of progesterone in human serum by isotope dilution liquid chromatography-tandem mass spectrometry and comparison with the commercial chemiluminescence immunoassay.

Progesterone is one of the steroid hormones. The hormone is especially important in preparing the uterus for the implantation of the blastocyst and in maintaining pregnancy. Its concentration in serum is measured to determine ovarian function and to predict early pregnancy. The progesterone concentration is also important for in-vitro fertilization and embryo-transfer outcomes. We have established isotope dilution liquid chromatography-tandem mass spectrometry as a primary method for the measurement of progesterone in human serum. Progesterone and its isotopic analogue, progesterone-(13)C(2), in serum were monitored at mass transitions of m/z 315.2/109.2 and 317.2/111.2 respectively in multiple-reaction monitoring (MRM) mode with electrospray positive ionization. For validation of the method, progesterone in a National Institute of Standards and Technology standard reference material (NIST SRM) was measured, and the measured results were in good agreement with the reference values within the uncertainty. On the basis of the established method, progesterone certified reference material (CRM) was developed in this work. The certified value was (1.41 ± 0.036) µg kg(-1). The repeatability of 1.1% and reproducibility of 0.14% showed that ID LC-MS-MS is a reliable and reproducible method. The expanded uncertainty for the measurement of progesterone in the CRM was approximately 2.6% within 95% confidence limits. The detection limit of progesterone was approximately 0.6 µg kg(-1). The progesterone CRMs were distributed to representative clinical laboratories in the Republic of Korea for comparison with the chemiluminescence immunoassay (CLIA), which is the most sensitive immunoassay method. The results from the comparison showed quite a large bias among the participating laboratories. This implies that the CRM is a very important material for establishment of traceability to its practical use.

Development of a candidate reference method for determination of tyrosine in serum by isotope dilution liquid chromatography-tandem mass spectrometry.

An isotope dilution liquid chromatography/tandem mass spectrometry is proposed as a reference method to determine the level of tyrosine in human serum. The advantages of this method include simple sample preparation without derivatization, the selective detection of compounds of interest in complex matrices, and the use of an isotopically labeled analogue as an internal standard. Tyrosine and its isotopically labeled analogue were monitored at a transfer m/z of 182.1/136.2 and 188.1/142.2, corresponding to [M+H]+/[M+H-HCOOH]+ in a multiple reaction monitoring mode. The expanded uncertainty for the measurement of tyrosine in the serum was approximately 0.95% within a 95% confidence level. For the verification of this method, a standard reference material with a certified value was analyzed. The analyzed result was in good agreement with the certified value. The isotope dilution liquid chromatography/tandem mass spectrometry result of the human serum was also compared with results obtained from clinical laboratories, and showed inconsistent results. These inconsistent results suggest that standards certified by the proposed reference method are required in order to improve measurement reliability in clinical fields.

Determination of phenylalanine in human serum by isotope dilution liquid chromatography/tandem mass spectrometry.

Isotope dilution liquid chromatography/tandem mass spectrometry (ID-LC/MS/MS) has been developed as a candidate reference method to determine the level of phenylalanine in human serum. The advantages of this method include a simple sample preparation without derivatization, selective detection of analytes, and the use of an isotopic analogue as an internal standard. Phenylalanine and its isotopic analogue, phenylalanine-ring-(13)C(6), were monitored at the transitions m/z 166.2/120.2 and 172.2/126.2 in the multiple-reaction monitoring (MRM) mode, respectively. The expanded uncertainty of the measurement result of phenylalanine in the serum was approximately 1.2% within a 95% confidence level. A standard reference material, with a certified value of phenylalanine, was analyzed in order to verify this method. The result obtained by the ID-LC/MS/MS method differed somewhat from the certified value, but agreed well with the gravimetric value. The measurement result of phenylalanine in serum by ID-LC/MS/MS was compared with the results from the commercial HPLC method, which was carried out in clinics. The results from the commercial HPLC method showed inconsistent results with each other. The busted results from the commercial HPLC method suggest that it should be possible to trace the results of the commercial fields to well-characterized reference materials or methods.