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Technical Briefs |
1 NIST, Analytical Chemistry Division, Gaithersburg, MD 20899-8392
aauthor for correspondence: fax 301-977-0685, e-mail sam.margolis{at}nist.gov
Ascorbic acid (AA) is one of the most important water-soluble antioxidants in biological systems. It is believed to be essential for scavenging free radicals, thereby reducing the mutation rate of DNA and preventing the development of diseases associated with gene mutations (1). AA plays an integral role in the synthesis of connective tissue and reduces biochemical cofactors and substrates (1). Serum concentrations of AA reflect the availability of AA for biochemical processes in humans and investigated animals (1). Our laboratory has been studying the precision and accuracy of serum AA concentration measurements performed by the medical research community (2). We have prepared several stable reference materials, as well as the stable Standard Reference Material (SRM) 970, Ascorbic Acid in Human Serum, for evaluating laboratory performance in the measurement of AA in human serum (2). We have distributed these materials in several international collaborative studies. These studies indicated that the interlaboratory precision is poorer than expected (relative SD, 10–30%). However, our laboratory studies on SRM 970 and other standard materials indicate that it is possible to routinely measure AA accurately with a high degree of precision (2).
Recently, we observed a significant decrease in the concentration of AA in our calibration solutions during the course of several experiments. We now document that the inside surface of glassware may contain materials that measurably degrade AA within a short period of time (<24 h). This is particularly true for autosampler vials used to hold samples for chromatography. This degradation may also occur in tubes and vials used in the collection and processing of blood and serum samples. It may well be that this type of degradation accounts for a substantial amount of the observed interlaboratory variation.
We evaluated nine different lots of vials (12 x 32 mm) from five different suppliers (Varian Analytical Instruments; Agilent Technologies; Thomson Instrument Co.; Alltech Associates; Chromacol). Two lots of vials were clear glass, and the remaining lots were amber glass. One lot of amber vials was silanized. A 7.39-mL (2-dram) vial and a borosilicate culture tube (Fisher Scientific) were also examined. The test solutions consisted of one or more of three different concentrations of AA (
0.19, 0.38, and 0.76 mg/L) dissolved in acetonitrile–water (3:1 by volume) containing 1 g/L dithiothreitol (DTT). These test solutions were stored in borosilicate flasks covered with aluminum foil. Each sample was analyzed by liquid chromatography (LC) on a Capcel Pak amine column (Phenomenex) as described previously (3) using electrochemical (EC) and ultraviolet (UV) detectors. Several cleaning procedures for reducing the degradation of AA were examined. The vials were washed by exposing them to the following solutions or groups of solutions for a period of 30 min each: (a) methanol–water (9:1 by volume) saturated with EDTA; (b) acetonitrile–water (3:1 by volume) containing 1 g/L DTT; (c) 0.5 mol/L NaOH followed by a distilled water wash, then 1 mol/L HCl followed by a distilled water wash; (d) distilled water followed by heating for 18 h in an annealing oven at 500 °C; (e) 0.1 mol/L nitric acid followed by distilled water wash; (f) 1 mol/L HCl followed by a distilled water wash. The effect of storing samples in vials at 22 °C and 4 °C was also evaluated.
Typical EC and UV chromatograms of an AA solution at zero time are illustrated in Fig. 1, A and C
, whereas Fig. 1, B and D
, shows similar chromatograms for the same solutions after standing in an autosampler vial from vial lot 1 for 22 h at 21 °C. Two oxidizable products (EC detector) and several additional UV-absorbing products were formed. Similar results were observed at 3.5 h for the same vial lot (7% degradation at 0.76 mg/L and 50% at 0.19 mg/L). Table 1
illustrates the results of testing a variety of vials from different lots and illustrates the variation in the stability of AA in the different vials for 22 h at 21 °C. Very little degradation (<3%) was observed in vial lots 6 and 7 during the first 3.5 h, and these were used for all previous measurements made in our laboratory (2). Vial lots 1–5 were autosampler vials obtained from a single distributor, and the remaining vial lots, 6–9, were obtained from other distributors. Container 10 was a borosilicate culture tube, and container 11 was a 7.39-mL (2-dram) vial. At least two vials were used for every test condition. Significant degradation of the AA was observed in all of the vial lots that were examined (including the containers that were used for sample preparation, vial lots 10 and 11). In some vial lots the degradation of AA was much less than in others (Table 1
).
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We performed several different washing and cleaning studies to determine whether the degradation of AA could be caused by contaminants on the inner surface of the glass vials. Of the procedures evaluated for reducing AA degradation, washing with acetonitrile–water (3:1 by volume) containing DTT (1 g/L), washing with methanol–water (9:1 by volume) saturated with EDTA, silanization, and heating in an annealing oven at 500 °C for 18 h had very little effect (Table 1
). Conversely, the acid wash and the base–acid wash treatments were effective in reducing the degradation of the AA. Because the most effective treatment utilized an acid wash and because the other treatments were ineffective, we believe that the source of the AA degradation is one or more transition metals that catalyze the oxidation of AA. This degradation was observed with amber vials (vial lots 1,2, 4–6, 8, and 9) and, to a lesser extent, with clear autosampler vials (vial lots 3 and 7) and the sample-preparation tubes (vial lots 10 and 11). For some vials, the amount of AA degradation varied among the vials (vial lots 1, 5, 6, and 9,).
AA degradation can be eliminated most effectively by soaking the vials in 0.5 mol/L NaOH for 30 min, rinsing with distilled deionized water (conductivity, 
18 siemens), soaking in 1 mol/L HCl, and again rinsing with distilled deionized water. Table 1
illustrates that this process was effective in reducing AA degradation in all of the vials that we evaluated. AA stability was also improved by treatment with 0.1 mol/L nitric acid (data not shown). Treatment with HCl also led to improved AA stability for all but two types of vials tested. Washing the vials eliminated the degradation that was observed at 3.5 h as well as that at 24 h. Cooling the vials to 4 °C also appeared to slow the reaction.
The phenomenon of trace amounts of metals adhering to glass is well documented in inorganic analysis of trace elements. Washing with hydrochloric acid or nitric acid is the standard procedure for the removal of these types of metal contaminants (4)(5). The presence of transition metal cations in the oxidized state bound to the inner surface of the glassware could easily account for the oxidation of AA to dehydroascorbic acid and further degradation products in biological samples (6). Because tubes and vials are used at every stage of sample processing (i.e., collection, storage, preparation, and analysis), this type of phenomenon may account for a significant amount of the large interlaboratory variation seen in our collaborative studies (2). This phenomenon may occur with reduced biochemicals other than AA and could account for the instability and variability of measurements of other important reduced biochemicals such as folic acid vitamers, flavins, and nicotinic acid dinucleotides.
Acknowledgments
Certain commercial equipment, instruments, and materials are identified in this manuscript to specify adequately the experimental procedure. In no case does such identification imply the recommendation of NIST, nor does it imply that the equipment or material is necessarily the best for the purpose.
References
The following articles in journals at HighWire Press have cited this article:
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  L. F. McCoy, M. B. Bowen, M. Xu, H. Chen, and R. L. Schleicher Improved HPLC Assay for Measuring Serum Vitamin C with 1-Methyluric Acid Used as an Electrochemically Active Internal Standard Clin. Chem., June 1, 2005; 51(6): 1062 - 1064. [Full Text] [PDF]
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