Clinical Chemistry 47: 2157-2159, 2001;
(Clinical Chemistry. 2001;47:2157-2159.)
© 2001 American Association for Clinical Chemistry, Inc.
Critical Role of pH for Derivatization of Homocysteine with Benzofurazanes
Anna Rita Bonfigli1,
Gilberto Coppa2,
Ivano Testa3,
Anna Gambini2 and
Roberto Testa1a
1
Department of Gerontological Research, Diabetology Unit, Italian National Research Centres on Aging, Via Birarelli 8, I-60121 Ancona, Italy;
2
Department of Clinical Pathology, General Hospital of Ancona, Ancona, Italy;
3
Institute of Internal Medicine, University of Ancona, Ancona, Italy
aauthor for correspondence: 39-071-2206106, e-mail r.testa{at}inrca.it
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Introduction
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HPLC analysis combined with fluorescence detection is one of the most effective and widely used techniques for the specific determination of total homocysteine in plasma (tHCY) (1)(2)(3)(4). These methods often use benzofurazanes as derivatization reagents that are considered to be specific for the thiol group of HCY (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). The specificity of benzofurazanes for thiols should be questioned because benzofurazanes have been used as NH2 derivatization reagents (15)(16). Because HCY possesses both -SH and -NH2 groups, we asked whether the widely reported pH of sample derivatization (from 8 to 9.5) (8)(13)(14) ensures thiol-selective derivatization. We performed sample derivatizations at pHs of 7.5–10.5 and an HPLC method (8) for tHCY determination with some modifications.
HCY, cysteamine, cysteine, cysteinylglycine, 7-fluoro-2,1,3-benzoxadiazole-4-sulfonamide (ABD-F), tris-carboxyethyl-phosphine, and other common reagents for the HPLC assay of tHCY were purchased from Sigma. Acetonitrile, isopropyl alcohol, and water, all HPLC grade, were obtained from Merck. The blood samples for tHCY determination were collected into Vacutainer Tubes (Beckton Dickinson) containing citrate. Plasma was immediately separated by centrifugation at 1500g for 10 min at 4 °C.
Sample preparation was performed as follows: reduction with tris-carboxyethyl-phosphine, derivatization with ABD-F, and precipitation of proteins. The derivatization was performed before the precipitation to avoid the realkalinization step. To 50 µL of the same sample, we added 100 µL of 250 mmol/L borate buffer at pH 7.5, 8, 8.5, 9, 9.5, 10, and 10.5. We added an internal standard (40 µL of a 50 µmol/L cysteamine solution), and then 30 µL of tris-carboxyethyl-phosphine (9.4 mmol/L in the same borate buffers), followed by 30 µL of ABD-F (9.2 mmol/L in 1 mol/L ammonium hydroxyde at the same pH as the borate buffer used). Samples were incubated at 55 °C for 15 min and then cooled at room temperature. Protein was precipitated by adding 50 µL of 600 mL/L perchloric acid. Precipitated HCY-ABD-F derivatives are stable 24 h at room temperature, avoiding direct sunlight.
We injected 50 µL of the supernatant into the chromatographic system [307 Pump HPLC (Gilson), FP 821 Fluorometer (Jasco), HP 3395 Integrator (Hewlett-Packard), and a Genesis octadecylsilane C18 column (150 x 4.6 mm i.d.; 4-µm particle size; Jones Chromatography)]. Fluorescence detection used excitation at 385 nm and emission at 515 nm. The mobile phase was 5 mmol/L potassium dihydrogen phosphate buffer containing acetonitrile (30 mL/L) and isopropyl alcohol (30 mL/L), pH 1.9. Recoveries were 
100% (i.e., initial concentration, 9.1 µmol/L; HCY added, 5 µmol/L; within-day recovery, 99.7% ± 2.4%; n = 5), and the assay was linear to 500 µmol/L. Comparison with the fluorescence polarization immunoassay on an IMx analyzer (Abbott) showed the following results: yIMx=0.878xHPLC + 0.583; r = 0.878; n = 136.
Two chromatograms obtained with the same solution of HCY (200 µmol/L) after derivatization with ABD-F at different pHs are shown in Fig. 1
. At pH 10.5, only the -SH ABD derivative peak was present (Fig. 1B
), whereas at pH 7.5, two peaks were seen (Fig. 1A
), with an -NH2 ABD derivative peak at 6.5 min and the -SH ABD derivative peak at 9.9 min. In Fig. 1E
, the percentage of the peak areas of -NH2 ABD derivatives and the -SH ABD derivatives are plotted vs the pH of the ABD-F derivatization. A pH-dependent mirror curvilinear trend is present for both the derivatives.
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Figure 1. Chromatograms of a solution of HCY (200 µmol/L) after derivatization with ABD-F at pH 7.5 (A) and pH 10.5 (B), and of nonpathologic human plasma (5.6 µmol/L tHCY) after ABD-F derivatization at pH 7.5 (C) and 10.5 (D).
The effect of pH of the derivatization step on the formation of the ABD-HCY derivates is reported in panel E. The peaks in the chromatograms are as follows: 1, -NH2 ABD-HCY; 2, -SH ABD-HCY; 3, cysteine; 4, cysteamine (internal standard); 5, cysteinylglycine. HPLC operating conditions were as follows: Genesis octadecylsilane C18 (4 µm) 150 x 4.6 mm column, eluted with 5 mmol/L potassium dihydrogenphosphate buffer containing 30 mL/L acetonitrile and 30 mL/L isopropyl alcohol, pH 1.9, at flow rate of 1.5 mL/min; fluorescence detection: excitation, 385 nm; emission, 515 nm.
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These data clearly highlight that the pH of derivatization has a critical role in this assay. Small variations of pH can change the results and increase imprecision. Unpredictable variation of plasma amines (e.g., from drugs, illnesses, or diet) may consume ABD-F, producing an underestimation of tHCY concentration. Taking into account these findings, we prefer to perform the ABD-F derivatization of tHCY at pH 10.5.
Chromatograms of the same plasma sample after derivatization at pH 7.5 and 10.5, respectively, are shown in Fig. 1, C and D
. Whereas Fig. 1D
shows a clean chromatogram with only cysteine, internal standard, cysteinylglycine, and tHCY without other interference, Fig. 1C
shows unknown peaks and higher apparent cysteine, presumably reflecting the overlapping of the -NH2 ABD-HCY peak.
We conclude that the pH of the derivatization of tHCY with benzofurazanes must be given particular attention in further attempts to standardize HPLC determination of tHCY.
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Acknowledgments
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We thank Dr. David Massey for support in the preparation and correction of the manuscript.
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