Simple, Rapid, and Accurate RP-HPLC Method for Determination of Cystine in Human Urine after Derivatization with Dansyl

Chloride
2007, 65, 527532

Yan Wang1, Xue-Jun Kang1,2,&, Wei-Hong Ge3, Xi-Zhao Sun3, Jie Peng3
1 2

School of Public Health, Southeast University, Nanjing 210009, China Key Laboratory of Child Development and Learning Science, Ministry of Education, Research Center for Learning Science (Southeast University), Nanjing 210096, China; E-Mail: xjkang64@163.com Drum Tower Hospital Afliated to Nanjing University, Nanjing 210008, China

Received: 27 November 2006 / Revised: 30 January 2007 / Accepted: 31 January 2007 Online publication: 27 March 2007

Abstract
An accurate, simple, and sensitive reversed-phase high-performance liquid chromatographic method, with loratadine as internal standard (IS) and UV detection at 286 nm, has been developed for deterination of cystine in human urine. The major innovations of the method include use of acrylonitrile to protect cysteine from oxidization to cystine, separation of cysteine, as the dansyl derivative, from cystine, and use of isocratic elution instead of gradient elution to reduce the time and cost of serial analysis. The mobile phase was 0.05 M sodium acetate methanol, 35:65 (v/v), adjusted to pH 3.5 with 2.5 M citric acid, at a ow rate of 1.0 mL min)1. The retention times of cystine and the IS were 16.6 and 19.9 min, respectively. The limit of detection for cystine was 0.3 mg L)1. Extraction recovery of cystine was >85.6%. Intra-day and inter-day precision (RSD) for cystine were below 4.3 and 8.5%, respectively. There was no chromatographic interference from other a-amino acids present in mammalian proteins, or from other urine components. The calibration plot for the cystine derivative was linear in the range 1500 mg L)1 and the correlation coefcient was 0.9992. The method was validated appropriately and successfully used for determination of cystine in human urine.

Keywords
Column liquid chromatography Precolumn derivatization Cystine in human urine Loratadine and acrylonitrile

Introduction
Cystinuria is an autosomal-recessive disorder of amino acid transport aecting the renal tubules and gastrointestinal tract [1]. It is a kind of nephrolithiasis manifesting as cystine calculi formation.

Its incidence has been reported to be between 1 in 15,000 and 1 in 7,000 [2]. Although the incidence of cystine calculi is low, they are hard to treat extracorporeally and relapse often occurs. When a calculus forms, surgical intervention is unavoidable. Quantitative analysis of

cystine in urine is, therefore, very important in preventing calculus formation and relapse. A variety of methods have been used for analysis of cystine, including colorimetric methods [3, 4], high-performance liquid chromatography (HPLC) [59], capillary electrophoresis [10], liquid chromatographytandem mass spectrometry [11], capillary electrophoresis dynamic reaction cell inductively coupled plasma mass spectrometry [12], and reversed-phase high-performance liquid chromatography (RP-HPLC)electrospray ionization mass spectrometry [13]. Although several HPLC methods are currently available for measuring cystine, none is satisfactory, because cysteine is susceptible to oxidization during sampling and sample-processing procedures. Hammermeister reported a method in which iodoacetic acid was used to protect the SH group of cysteine [13], but the reaction conditions had to be strictly controlled and information about conversion efciency was not given. Quantitative methods for clinical analysis of cystine in urine have been reported and include use of an amino acid analyzer, thin-layer chromatography (TLC), and a variety of colorimetric methods [14]. The objective of the work discussed in this paper was to establish a fully validated high-performance liquid chromatographic method with ultraviolet detection (HPLCUV) for determination of cystine, including use of acrylonitrile

Original DOI: 10.1365/s10337-007-0210-1 0009-5893/07/05

Chromatographia 2007, 65, May (No. 9/10) 2007 Friedr. Vieweg & Sohn Verlag/GWV Fachverlage GmbH

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to protect the SH group of cysteine from oxidization, derivatization of the analyte with dansyl chloride (DNS-Cl), and extraction of the dansyl derivative with dichloromethaneisopropyl alcohol, 4:1 (v/v), then separation by RP-HPLC. The HPLCUV method reported in this paper is a simple, accurate procedure for determination of cystine in urine.

Chromatography
Chromatography was performed with a Shimadzu (Japan) series 200 IC pump and an SPD-10A UVvisible detector. Compounds were separated on a 150 mm 4.6 mm i.d., 5 lm particle, Shim-pack C18 column (Shimadzu). All analysis was conducted in isocratic mode. The mobile phase was 35:65 (v/v) 0.05 M sodium acetatemethanol adjusted to pH 3.5 with 2.5 M citric acid; the ow rate was 1 mL min)1. Solutions and mobile phases were prepared immediately before use. The assay used UV detection at 286 nm. Peak-area measurement was used for quantication. Total run time was 21 min. All analysis was performed under the same conditions at room temperature.

Experimental
Chemicals and Reagents
DNS-Cl, cysteine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, glycine, and cystine were obtained from Sigma (St. Louis, MO, USA). Loratadine, used as internal standard (IS), was provided by China Pharmaceutical University. Methanol (HPLC-grade) was purchased from Shucheng Chemical Factory (Shandong, China). Potassium hydroxide, acetone, acrylonitrile, sodium bicarbonate, dichloromethane, isopropanol, diethyl ether, ethyl acetate, butanol, and iodoacetic acid were obtained from Shanghai Reagent Factory (Shanghai, China). All chemicals were analytical-reagent grade. Doubledistilled deionized water was used throughout the study.

Sampling Procedure
Urine samples were stored at )20 C and left to thaw at room temperature before analysis. Samples and standards (500 lL) were saturated with sodium bicarbonate in a centrifuge tube then 5 lL acrylonitrile was added. After capping the tube the samples were vortex mixed (30 s), ultrasonicated (10 min), and incubated at room temperature for 20 min. After addition of 250 lL 20 g L)1 DNS-Cl the samples were vortex mixed (30 s) and the mixture was reacted in the dark at 80 C in a water bath for 30 min. For extraction the mixture was centrifuged at 10,000 g for 5 min and supernatant (400 lL) was transferred to another centrifuge tube. Acetic acid (50 lL) was added to adjust the pH to 5.0, 10 lL IS working solution was added, and the solution was mixed vigorously for 30 s. Dichloromethaneisopropyl alcohol (4:1, v/v; 2.5 mL) was added as the extracting solvent and, after mixing vigorously for 60 s, the mixture was centrifuged (10,000 g for 5 min). The upper layer was removed and evaporated to dryness under a gentle stream of nitrogen. The residue was dissolved in 100 lL mobile phase and 20 lL of this solution was analyzed by HPLC.

furnish concentrations of 1, 10, 200, 300, and 500 mg L)1 cystine. The protecting reaction and derivatization were then performed as described above. A calibration plot was constructed by plotting the cystine-to-IS peak-area ratio against the concentration of cystine, and the linear regression equation was calculated. Extraction recovery was calculated for urine samples spiked at 5, 100, and 500 mg L)1 (n = 6 for each) by comparing the peak-area ratios obtained from extracted samples with those obtained by direct injection of the corresponding unextracted standards. The intra-day and inter-day precision and accuracy of the method were evaluated for urine samples spiked with cystine (n = 6) at concentrations of 5, 100, and 500 mg L)1. Inter-day precision was evaluated on six successive days. Imprecision is reported as the relative standard deviation (RSD) of analyte concentration.

Results and Discussion

Optimization of the Derivatization Conditions
Cystine and cysteine neither uoresce nor absorb strongly in the UVvisible region. If HPLCUV is used for quantication of cystine, derivatization is needed. To improve the sensitivity and accuracy of cystine analysis the derivatization conditions temperature, time, pH, and concentration of DNS-Cl were optimized. A standard solution (500 mg L)1) of cystine was used as a model sample. The highest yield of DNS-Cl derivative was obtained when the concentration of DNS-Cl was 20 g L)1. Different temperatures and reaction times were examined to nd the optimum conditions for derivatization of cystine; 80 C and 30 min were used throughout this study. The reaction usually requires an alkaline pH of 9.5 [15]. For the pH ranges examined saturation of the solution with sodium bicarbonate was selected. The results obtained are shown in Fig. 1.

Standard Solutions
An aqueous stock solution of cysteine (1 g L)1) was prepared by dissolving 10 mg cysteine in 10 mL double-distilled water. Cystine stock solution (1 g L)1) was prepared by rst dissolving 10 mg in 160 lL potassium hydroxide (5 M), because of its low solubility in acidic and neutral media, then diluting to 10 mL with double-distilled water. These solutions were diluted daily to the working concentrations with double-distilled water. The IS stock solution was prepared in methanol at a concentration of 1 g L)1 and diluted daily to the working concentration (0.25 g L)1) with water. All solutions were stored at 4 C in the dark. DNS-Cl solution was prepared immediately before derivatization by dissolving 200 mg DNS-Cl in 10 mL acetone.

Optimization of the Extraction Conditions Validation of the Assay

Standard stock solution was added to double-distilled deionized water to Chromatographia 2007, 65, May (No. 9/10) Liquidliquid extraction was used for sample preparation in this investigation. An immiscible organic phase was added Original

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Fig. 1. Eect on derivatization yield of a concentration of dansyl chloride, b temperature, c reaction time, and d pH

to the aqueous solution of dansyl cystine and this mixture was then shaken, resulting in transfer of most of the dansyl cystine and IS into the organic phase. The best extracting solvent must be selected for ecient liquidliquid extraction. By comparing the extraction eciency of dichloromethaneisopropyl alcohol, diethyl ether, ethyl acetate, and butanol we conrmed that extraction eciency was highest with dichloromethaneisopropyl alcohol, 4:1, and lowest for ethyl acetate. Butanol was not well separated from the aqueous solution. Subsequent investigation of the eect of pH showed that pH 5 resulted in the optimum extraction eciency.

RSH H2 C=CHCN
OH

RS--CH2 --CH2 --CN !

Protecting Reaction
Protection of the SH group of cysteine with iodoacetic acid has been reported [13], but the operation is complicated, time-consuming, and no information about conversion efciency is available. In our experiment, acrylonitrile was reacted with cysteine, in the presence of small amounts of alkali as condensing agent, to form the thioether [16]. Original

Cysteine could not be converted into cystine, so cysteine and cystine could be eectively separated within 18 min with good peak resolution, sharpness, symmetry, and reproducibility. Accurate quantication of cystine was possible by use of this method. We also investigated the volume of acrylonitrile needed; 5 lL was sucient for 500 lL solution containing 500 mg L)1 cysteine. The effect of temperature on conversion efciency was studied in the range 2060 C for different periods of time. It was found that 20 min was sufcient for conversion at room temperature (20 C). To determine conversion eciency, cysteine solutions were diluted to concentrations of 5, 100, and 500 mg L)1 (n = 6 for each) with double-distilled water. The protecting reaction was repeated three times as described above at each concentration; other solutions were analyzed without addition of acrylonitrile. Ratios of cystine peak areas (reduced peak area without addition of acrylonitrile divided by peak area with addition of acrylonitrile) were used to calculate the efciency of conversion of Chromatographia 2007, 65, May (No. 9/10)

cysteine (Fig. 2). The efciencies of conversion were 97.1, 96.4, and 96.3% for cysteine at concentrations of 5, 100, and 500 mg L)1, respectively. The stability of the thioether (product of reaction of cysteine with acrylonitrile) was determined by periodic analysis of a standard solution stored at 4 C. No signicant change in peak areas was observed after 7 days.

Internal Standard
Changes can occur during pretreatment and determination, so it is important to monitor this by use a suitable IS. If an amino acid was used as the IS it was possible assay variability would be introduced, because of dierent rates of derivatization. Loratadine was selected as IS because of its suitable retention time and stability; its use enabled monitoring of extraction, injection volume, column performance, and detector response. Under our chromatographic conditions it was not co-eluted with any other peak.

Validation of the Assay

Selectivity

Typical chromatograms obtained from analyte-free blank, urine spiked with

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was the analyte-to-IS peak-area ratio and x was the concentration of the analyte. The correlation coefcient (r) for cystine was 0.9992 over the concentration range used. The standard deviations of the slope and intercept of the calibration plot were 0.0005 and 0.1417, respectively.
Limits of Detection and Quantitation

The limit of detection (LOD) was calculated as three times the standard deviation for six replicate analyses of procedure blanks. The limit of quantitation (LOQ) was established at a signal-to-noise ratio of 10:1 for six replicate analyses of procedure blanks. The LOD and LOQ for cystine were 0.3 and 1.0 mg L)1, respectively.
Accuracy and Precision

The precision of the method was estimated by performing a series of replicate analyses of urine spiked with cystine. The intra-day precision (n = 6), calculated from the peak-area ratios, was within the range 2.34.3%. The inter-day precision (n = 6), calculated from the peak-area ratios, ranged from 3.1 to 8.5%. The relative recovery ranged from 97.0 to 108.5% (n = 6). The results are shown in Table 1.
Extraction Recovery

Extraction recovery was determined for urine samples spiked with 5, 100, and 500 mg L)1 cystine (n = 6) by comparing the peak-area ratios obtained from extracted samples with those obtained by direct injection of the corresponding unextracted standards. The extraction recoveries were 102.2, 98.4, and 85.6% for urine spiked with 5, 100, and 500 mg L)1, respectively.
Stability
Fig. 2. High-performance liquid chromatograms obtained from cystine, cysteine, and 19 a-amino acids: a analyte-free blank; b cystine (CSSC) and cysteine (C) standards; c urine spiked with cystine (CSSC) and cysteine (C) standards; d 19 a-amino acids; e cysteine (C) without addition of acrylonitrile; f cysteine (C) with addition of acrylonitrile

standard, and a standard solution are shown in Fig. 2. The retention times of the cystine adduct and the IS were 16.9 and 19.9 min, respectively. No chromatographic interferences from the urine were observed. Other a-amino acids present in mammalian proteins did not interfere.

Linearity and Range

The cystine-to-IS peak-area ratio for standard solutions was a linear function of analyte concentration over the range 1500 mg L)1; the linear regression equation for the calibration plot for cystine was y = 0.0077x ) 0.0222, where y

Stability was determined by storing standard and sample solutions, after sample preparation, at 4 C and analyzing the solutions after 24 h, 7 days, and 30 days. The results indicated the dansyl derivative of cystine was very stable at 4 C for 30 days; the stability of standard solutions and sample solutions before sample preparation were determined by storing the solutions at )4 and )20 C and analyzing the solutions after 24 h and 7 days. The results indicated cystine was stable for at least 7 days. The method

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Table 1. Intra-day and inter-day precision and accuracy of the assay for cystine (n = 6)
Spiked amount (mg L)1) Intra-day 5 100 500 Inter-day 5 100 500 Assay value (mean SD, mg L)1) Coecient of variation (%) Accuracy (%)

Table 2. Results from system-suitability study

Characteristic Resolution Tailing factor Theoretical plates Repeatability Value 7.34 1.1 5231 0.001 (n = 5)

4.8 0.2 100.4 2.3 523.3 22.6 4.8 0.41 97.6 3.1 542.7 29.3

3.1 2.3 4.3 8.5 3.1 5.4

)3.0 0.4 4.7 )3.0 )2.4 8.5

was stability-indicating and there was no interference from degradation products.

Ruggedness

The ruggedness of the method was evaluated by making small changes to the method conditions. The conditions chosen for this study were the detection wavelength (nm), mobile-phase ow (mL min)1), and mobile-phase composition. When the ow rate was 10% higher or lower than the normal ow rate no signicant change in peak area was observed. When wavelengths of 280, 286, 290 nm were tested there was no signicant change in peak area. It was found that mobile phase composition had very large effect on peak retention time and separation.
System Suitability

Fig. 3. High-performance liquid chromatogram obtained from urine sample

To verify system performance the systemsuitability data, peak resolution, tailing factor, theoretical plate number, and repeatability were calculated using 100 mg L)1 standard solution (Table 2).

Chromatographic and Detection Conditions

The chromatographic conditions, especially mobile phase composition and pH, were optimized by means of several trials to reduce analysis time and achieve symmetrical peak shapes for the analyte. Mobile phases prepared from mixtures of 0.05 M sodium acetate and methanol in the ratios 75:25 to 50:50 (v/v) were tested and it was found that the mixture containing 35% 0.05 M sodium acetate and 65% methanol (v/v) achieved our purpose. The effect of mobile phase pH was then studied. It was found that pH had very large effect on peak retention time; a pH of 3.5 was selected because it resulted Original

in reasonable retention times and a symmetric peak for cystine. The best response was achieved at a detection wavelength of 286 nm. Cystine and the IS were well separated from other peaks in urine under these chromatographic conditions. Figure 2b and c show representative chromatograms obtained from a standard solution and from a urine sample spiked with standards. The order of elution was cysteine (4.2 min), then cystine (16.6 min), then the IS (19.9 min). C and CSSC are the products of reaction of cysteine and cystine, respectively, with DNS-Cl. The chromatograms show only one product (the di-dansyl product) obtained from cystine, because systemic peaks derived from hydrolysis of DNS-Cl co-eluted with the peak produced by mono-dansylation of cystine. This did not affect method reliability. The peak area obtained from the di-dansyl product of cystine was a linear function of analyte concentration over the range 1500 mg L)1. The validation data revealed that precision and accuracy were good, as already described. If acrylonitrile was not added, cysteine was converted to cystine during derivatization (Fig. 2e). Interfering peaks from urine always co-eluted with peak C, so cysteine was not quantied in this work. To determine whether other amino acids interfered with quantication of the target compound a mixture of 19 other a-amino acids (glycine alanine, valine, leucine, isoleucine, phenylalanine, proChromatographia 2007, 65, May (No. 9/10)

line, tryptophan, serine, tyrosine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine) was tested and no interference was observed (Fig. 2d).

Application
The assay procedure was used for analysis of urine from healthy persons. Amounts of cystine in urine from healthy volunteers were between 7.84 and 10.1 mg L)1 (3,7104,776 lmol mol)1 creatinine). These amounts are in excellent agreement with values previously reported for healthy persons [17]. A typical chromatogram obtained from urine is presented in Fig. 3.

Conclusions
The rapid and accurate method proposed in this paper is a convenient means of quantication of cystine in human urine. It is simpler than the HPLC method described previously, the run time is suitable (21 min), and 96.6% of the cysteine was protected from oxidization to the disulde. The validation data reveal the precision and accuracy are good. The validated method enables quantication of cystine over the range 1.0500 mg L)1 and the method may be useful for routine determination of large numbers of samples.

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Acknowledgments
This work was partially supported by a grant from Ministry of Education of China and Foundation of Nanjing Health Bureau (no. ZKM05047).

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