High-performance liquid chromatographic method for the determination of benzodiazepines in plasma or serum using the column-switching technique

A column-switching high-performance liquid chromatographic method for the simultaneous determination of five frequently prescribed benzodiazepines: clonazepam, diazepam, flunitrazepam, midazolam and oxazepam was developed. A 50-μl plasma sample was directly injected into a BioTrap 500 MS (hydrophobic polymer) column. After a washing step with a mixture of phosphate buffer and acetonitrile, the retained benzodiazepines were back-flushed into a reversed-phase (LiChrospher Select B C₈) column with a mobile phase of acetonitrile–phosphate buffer. The method showed excellent linearity from 50 to 1000 ng/ml for clonazepam, flunitrazepam and midazolam and from 50 to 5000 ng/ml for diazepam and oxazepam. The recoveries were around 98% for all the benzodiazepines studied. The relative standard deviation for between- and within-day assay was <20% for low concentrations close to the values of the limit of quantification and <4% for high concentrations. The procedure described is relatively simple and rapid because no off-line manipulation of the sample is required: the total analysis time is approximately 30 min.     

Assay of alpha-cysteine proteinase inhibitor in serum or plasma

A new proteinase inhibitor has recently been found in human serum or plasma which specifically inhibits cysteine proteinases such as ficin, papain, bromelain and cathepsin B. However, serum contains alpha 2-macroglobulin which also inhibits these cysteine proteinases and, consequently, interferes with the assay of the new alpha-cysteine proteinase inhibitor. Therefore, assay of the inhibitor in serum has not been established previously. In the present method, the alpha 2-macroglobulin is inactivated by preincubating the serum in methylamine solution at 55 degrees C, while the alpha-cysteine proteinase inhibitor retains its activity. The inhibitory power against cysteine proteinases is found to be due mainly to this protein in human serum. This inhibitor is also found in mammals such as cows, pigs and rats. Vitamin E deficient rats show a very high inhibitor level. Therefore, the present method will enable us to investigate the relation between diseases and the activity of the alpha-cysteine proteinase inhibitor. Also, this method is simple and inexpensive. The necessary amount of serum is only 10 microliter.     

A microliter method for the gas chromatographic determination of long-chain non-esterified fatty acids in human serum or plasma

Non-esterified fatty acids (NEFA) from C₁₂ to C₂₄ are assayed in human serum or plasma in a four-step procedure: extraction, volume reduction, methylation and gas chromatography. NEFA are extracted with chloroform—heptane—methanol from 50–100 μl of serum or plasma buffered with phosphate. After adding ethyl acetate the volume of the extract is reduced under partial reflux to 5–7 μl. Potassium carbonate, methyl iodide and a crown ether are added to the dry concentrate and the NEFA are selectively methylated with a yield of 100% by heating in a microrefluxer for 10 min. Gas chromatography is carried out with 1 μl of the reaction mixture on a packed column by temperature-programmed operation. Thirteen individual fatty acids are determined in sera of normal adults. The coefficients of variation for 24 determinations of a pooled serum were 2.7% for the total NEFA content and 3–10% for most of the individual NEFA.     

Long-term stability of biomarkers of the iron status in human serum and plasma

Abstract

Context: In epidemiological research, it is very important to test the stability of biomarkers as function of both storage time and temperature.

Objective: In this study, the stability of biomarkers of the iron status was tested up to 1 year of storage.

Materials and methods: The biomarkers include total iron, unsaturated iron binding capacity, ferritin, transferrin, soluble transferrin receptor, ceruloplasmin and haptoglobin.

Results: The concentrations of all biomarkers tested remain constant upon storage at ?20, ?70 and ?196?°C.

Conclusion: All biomarkers of the iron status were stable at the temperatures tested for 1 year.     

Refrigeration of blood samples prior to separation is essential for the accurate determination of plasma or serum zinc concentrations

An evaluation of refrigeration (7°C) to prevent falsely high plasma or serum zinc concentrations owing to elapsed time between blood collection and centrifugation was performed. At room temperature (23°C), both plasma and serum zinc concentrations increased significantly, if blood samples were stored uncentrifuged. Plasma zinc concentrations increased 6.3% at 1 h and 40.7% at 24 h, whereas serum zinc concentrations increased only 0.9% at 1 h and 12.5% at 24 h at room temperature. When blood samples were stored uncentrifuged in the refrigerator for up to 24 h, there were no significant increases in zinc concentrations in either plasma or serum. These findings suggest that plasma or serum separation should be performed immediately after blood drawing to obtain accurate zinc concentrations, and if this is not feasible, the samples should be immediately refrigerated and separation performed within eight hours.