Question: Literature search problem: Human serum albumin (HSA) is an important protein ingredient in cryopreservation media used in procedures such as in vitro fertilization. Search the literature for a high-performance liquid chromatography method for the determination of human serum albumin and the stabilizer N-acetyl tryptophan in medical devices.

(a) Give the citation (authors, title, journal name, year, volume, pages) for the research paper that fits the criteria of this analysis.

(b) What alternative methods could be used for analysis of human serum albumin?

(c) What type of analytical column is used for the separation?

(d) How long was the gradient? How long were the additional wash and equilibration steps within the gradient method?

(e) What parameters were assessed in the method validation?

(f) Why were particles with 300 Å pores used?

Human serum albumin (HSA) is the amplest plasma protein generated in the liver. It plays a major role in several physiological processes, which include hormone transport, ion transport, maintenance of the osmotic pressure, fatty acid binding, blood pH buffering, etc. It is extensively used for several medical purposes also. HSA is used for treating several medical conditions like kidney disorder treatments, hypoproteinemia, burns, hepatic failure, and also in maintenance and circulation of blood volumes in case of (hypovolemic) shock.

Its nutritional, anti-oxidant, and cryoprotective properties make it an important ingredient in culture, freezing, and thawing media. These are used for assisted reproductive techniques (ART), including in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) procedures, and cryopreservation of gametes and embryos. Due to the application of HSA in various fields, it is very much needed to determine the accurate concentration of HAS in a sample.

(a)Citation:

Authors: Frank Eertmans, Veerle Bogaert and Barbara Puype

Title: Development and validation of a high-performance liquid chromatography (HPLC) method for the determination of human serum albumin (HSA) in medical devices

Journal name: Analytical Methods

Year: 2011

Volume: 3

Pages: 1296-1302

(b) The alternative methods that could be used for the analysis of human serum albumin are colorimetric BCG (bromocresol green) and BCP (bromocresol purple) tests, biuret method, fluorometric tests, electrophoretic technique, immunological test, etc. But there are several disadvantages like accuracy problems, sensitivity problem, etc., regarding these tests.

In this case, a reverse phase high-performance liquid chromatographic (RHPLC) method was used to analyze human serum albumin. A properly developed HPLC possesses several advantages like short analysis time, high resolution, high sensitivity, reproducibility, accuracy, and automation possibilities. For the determination of human serum albumin, several HPLC methods like size exclusion, ion exchange, reverse phase (RP) separation, etc., are available.

(c) The analytical column used was a thermoregulated VYDAC 214TP C4 column (250 mm × 4.6 mm i.d.;), consisting of polymerically bonded, end-capped n-butyl-coated silica particles (5 mm) with a pore diameter of 300 Ǻ with a guard column, containing identical C4 material as the former.

(d) The gradient was 20 min long with eight equilibration steps. The mobile phase A being a 0.1% TFA (v/v) aqueous solution, and mobile phase B is 0.1% TFA (v/v) in 100% acetonitrile (ACN), was used for chromatographic separation. A table regarding the gradient elution scheme of chromatographic separation from literature is depicted below

(e) In the method of validation, the parameters assessed in this literature were peak identification, determination of the linearity and range, precision (repeatability and intermediate precision), accuracy, robustness, specificity (LOD and LOQ), and sensitivity.

This method is able to reliably quantify HSA concentration in the range of 0.4–25 mg ml^-1, with a LOD (Limit of Detection) and LLOQ (Lower Limit of Quantification) of 0.128 and 0.386 mg ml^-1, respectively, as calculated using the slope method.

(f) Human serum albumin is a relatively large protein (66–67 kDa) compared to others; therefore, it was chosen to fill the C4 column with 300 Ǻ pore size silica particles.