HPLC Qualitative Amino Acid Analysis in the Clinical Laboratories

HPLC Qualitative Amino Acid Analysis in the Clinical Laboratories JOE C. RUTLEDGE, M.D. AND JACK RUDY, M.S. The authors have developed a rapid system for qualitative amino acid analysis by high-pressure liquid chromatography for use in the clinical laboratory. Reverse-phase, linear gradient, highpressure liquid chromatography of stable dansyl chloride derivatives is accomplished with a C-18 column over 30 minutes, with ultraviolet detection. The resulting chromatograms are compared with template chromatograms that reflect high normal levels of serum and urine amino acids. Large elevations of amino acids associated with inborn errors of metabolism are easily pinpointed, with identification of specific amino acids made by the relative retention times. This technic makes available a rapid and specific qualitative method for investigation of aminoacidopathies and uses versatile and standard liquid chromatography equipment. (Key words: Amino acid analysis; HPLC; Aminoacidopathies; Inborn errors of metabolism; Dansyl chloride) Am J Clin Pathol 1987; 87: 614-618 AMINO ACID ANALYSIS in the clinical laboratory has served to support the diagnosis of aminoacidopathies and the titration of amino acid levels in response to therapeutic intervention, for example, diet and/or mega-vitamin therapy. The major utility for amino acid analysis has been in pediatrics and the inborn areas of intermediary metabolism, which, because of specific enzymatic defects, are associated with extraordinary elevations of certain amino acids. 9 Recently, expanded uses for amino acid analysis have been introduced into both pediatric and adult clinical medicine, including the prognosis of severe liver disease, 8 the treatment of hepatic encephalopathy with branch chain amino acid solutions, 5 and the monitoring of parenteral nutrition therapy.' In both the diagnostic uses of amino acid analysis and the uses centering around therapeutic monitoring, a rapid result is often necessitated. As the clinical usefulness diversifies, rapid, simple, and inexpensive analysis will be necessary. Presently, qualitative amino acid analysis depends upon a battery of spot urine tests, one- or two-dimensional paper or thin-layer chromatography, high-voltage electrophoresis, occasionally, gas liquid chromatography, or dedicated ion exchange amino acid analyzers. 3 The qualitative Received May 29, 1986; received revised manuscript and accepted for publication July 28, 1986. Presented in part at the Society for Pediatric Pathology Meeting, Toronto, March 1985. Address reprint requests to Dr. Rutledge: Pathology Department, Children's Medical Center, 1935 Motor Street, Dallas, Texas 75235. Department of Pathology, Children's Medical Center, and the University of Texas Health Science Center at Dallas, Dallas, Texas tests lack specificity and involve subjective interpretation, necessitating a degree of experience built on close clinical pathologic correlation. The spot tests, while rapid, are nonspecific and, as in the case of ferric chloride, react with a diversity of compounds. A positive spot test, moreover, provides no information concerning the elevations of individual constituents present. Spot tests are usually run on urine and, as such, are susceptible to dilution-concentration effects by urine flow and do not readily reflect the status of the plasma amino acids, which are more important, in the detection and monitoring of all but the renal tubular defects. High-voltage electrophoresis of amino acids, requiring bulky equipment, provides for some separation and hence, semiquantitative identification of elevations. Paper and thin-layer chromatography, both one and two dimensional, can be achieved with a small capital equipment investment, if the laboratory already has appropriate ventilation. Both provide for separations of amino acids to allow a reasonable turnaround time for the semiquantitative reporting. 7 These chromatography technics are susceptible to interferences and require a degree of experience and skill in interpreting size and color of spots. Moreover, large quantities of one amino acid may mask elevated quantities of those with similar migration rates. Amino acids may be derivatized and separated by gas liquid chromatography. Many of the derivatization procedures are tedious 10 ; and, presently, gas liquid chromatography has had decreased utility in clinical chemistry laboratories. The dedicated amino acid analyzers are expensive and are rarely justified on the basis of clinical specimens alone. High-pressure liquid chromatography (HPLC) has been used for more than a decade by protein chemists to study the amino acid composition of protein hydrolyzates. 13 Drawing from that data, we sought to develop a system for qualitative amino acid analysis that uses the rapid separatory capabilities of HPLC to produce chromatograms used to provide rapid, specific, semiquantitative information for diagnostic purposes. Many of the previously 614

Vol. 87 No. 5 HPLC AMINO ACID ANALYSIS 615 reported HPLC procedures performed poorly with physiologic fluids, or the derivatization procedure had drawbacks retarding their usefulness in the clinical laboratory. We selected the dansyl chloride reaction as the most adaptable to our needs, optimized the reaction, and evaluated the procedure in comparison with a reference method, for its utility in our patient population, and for acceptability in our laboratory. Methods A rapid HPLC analysis for amino acids was developed and verified with 52 specimens simultaneously quantitated by a Beckman Model 119 ion-exchange, dedicated amino acid analyzer. Samples used for comparison were those submitted to the laboratory for quantitative amino acid analysis by ion exchange chromatography. The samples originated from a patient population being screened routinely for aminoacidopathies, patients suspected of having aminoacidopathies, and patients with previously diagnosed aminoacidopathies. No attempt was made to selectively analyze specimens in any one of these groups. The performance was monitored for one year after institution of the analysis. The retention times of amino acids standards were determined, as was the completeness of derivatization and the instrument response. Equipment A Perkin-Elmer series 400 liquid chromatograph was equipped with a guard column, preceeding a 15-cm, 5 Mm, C-18 DuPont analytical column encased in a water bath at 40 C (+ or - 1 C). A Perkin-Elmer LC-75 ultraviolet detector at 254 nm was connected to a Sigma- 15 data console. Sample Preparation Proteins were precipitated by mixing 100 nl of the specimen with 250 pl of a 100 /imol/l tryptamine/acetonitrile solution. The solution was centrifuged after vortexing for 30 seconds and 250 fil of supernatant was derivatized. Urine specimens were handled in a similar fashion, except when the urine creatinine exceeded 25 mg/dl (2,200 Mmol/L); the volume for analysis was adjusted to (25/urine creatinine) X0.10. Derivatization Two hundred fifty microliters of prepared specimen was mixed with 230 nl of 0.3% lithium carbonate, ph 9.5, and 20 nl of dansyl chloride reagent, prepared by dissolving 50 mg of dansyl chloride in 1 ml of acetonitrile (stored at -4 C and protected from light). After reacting in the dark at room temperature for 30-60 minutes, a clear supernatant was removed and reserved for injection. Chromatography Reverse-phase chromatography was accomplished with a linear gradient of the acetonitrile and a ph 4.0, 0.003 mol/l sodium phosphate buffer. After equilibration at 15% (v/v) acetonitrile for 5 minutes, 25 nl of serum or 10 /xl of urine was injected with an attenuation of 4. After 1 minute at 15% (v/v) acetonitrile, a 15-80% (v/v) acetonitrile gradient was run over 29 minutes. The concentration of acetonitrile was held steady for 3 minutes. Before the 5-minute equalisation time, the acetonitrile is returned to 15% (v/v) via a linear gradient over 2 minutes. Data Reduction The chromatogram was compared with a serum or urine template standard (Figs. \A and B) (chromatograms prepared under the same conditions, utilizing concentrations of the amino acids near generalized upper limit of normal). Peaks exceeding the heights of those on the templates, and having relative retention times corresponding to amino acids, were interpreted as elevations of particular amino acids. The specificity and degree of reporting depends on the pattern of amino acid elevations detected. Most amino acids can be readily quantitated by comparison with peak heights of known standards. Standards and Controls The analysis was standardized using serum and urine template standards. The amino acids were dissolved in water at concentrations equivalent to the upper threefourths of the normal range of serum and the upper threefourths of the normal range of urine, presuming a urine creatinine of 25 mg/dl (2,200 jtmol/l). The appropriate standard template was run with each series of analyses. Sigma Acid, Base, Neutral Amino Acid Solution (Sigma Chemical Co., St. Louis, MO 63178) contained 38 amino acids at a concentration of 500 ^M/L; each was also run with each series of analyses, serving as a positive control. Results Representative chromatograms of the new method are presented in Figures \C-H. Thirty-two serum specimens were studied by qualitative and quantitative technics. Twenty-five had normal screens, and seven had abnormal screens. Twenty-six had normal quantitative amino acids, while six had abnormal quantitative amino acids, and there were two false positives and one false negative, yielding a sensitivity of 92% and a specificity of 93%. One false positive showed minor elevations of glutamine and aspartic acid, which were not confirmed on the amino acid analyzer, while the other showed elevations of glutamine, isolecine, hydroxylysine,

616 RUTLEDGE AND RUDY is A.J.C.P. May 1987 SERUM TEMPLATE Y \J PHENYLKETONURIA, SERUM NORMAL SERUM u DO U HH KK UA^AJ^J ARGINASE DEFICIENCY, SERUM NORMAL URINE J \u UIIL^/VJI^ FIG. 1. Peak identification and amino acid localization. A = phosphoserine; B = amino adipic acid; C = dansyl reaction products; D = glutamic acid; E = asparagine; F = glutamine; G = aspartic acid; H = serine; / = phosphoethanolamine; J = hydroxyproline; K = glycine; L = citrulline; M = taurine; N = alanine; O = threonine; P = arginine; Q = sarcosine; R = a-aminobutyric acid; S = /3-amino isobutyric acid; T = lysine; U = valine; V = leucine; W = phenylalanine; X = beta-alanine; Y = methionine; Z = isoleucine; AA = hydroxylysine; BB = 7-aminobutyric acid; CC = cystathionine; DD = ethanolamine; EE = tryptophan; FF - cystine; GG = homocystine; HH = histidine; // = 1-methylhistidine; JJ = 3- methylhistidine; KK = ornithine; LL = proline; MM = tyrosine; AW = argininosuccinic acid; 00 = tryptamine = internal standard; PP = dansyl chloride. A (upper, left). Serum template. B (upper, right). Urine template. C (center, left). Normal serum. D (center, right). Elevated phenylalanine. E (lower, left). Arginase deficiency. F (lower, right). Normal urine. Continued. histidine, ornithine, and tyrosine, which were not confirmed by amino acid analysis. The false negative screen was one in which an elevation of tyrosine at 240 jtmol/l (normal range 23-147) was not detected. In addition, on three separate occasions, the quantitative amino acids showed a marked elevation of cystine, which did not show by the HPLC technic. There is no clinical explanation for such an elevation, and, using additional technics, we were

vol. 87-No. 5 MAPLE SYRUP URINE DISEASE HPLC AMINO ACID ANALYSIS FANCONI SYNDROME URINE 617 t M FIG. 1. Continued. G (left). Maple syrup urine disease. H (right). Aminoaciduria from renal tubular defect. unable to confirm that the peaks seen on the amino acid analyzer, indeed, did represent cystine and treat it as a quantitative amino acid analysis false positive. Twenty urine specimens were compared with 13 normal screens confirmed by quantitative amino acid analysis and 7 abnormal screens likewise confirmed, yielding sensitivity and specificity for this group of 100%. Discussion HPLC equipment has found its place in many clinical chemistry laboratories because it provides a system for analysis of a wide variety of compounds for which commercial instrumentation and reagents are not available. Our amino acid analysis screening system uses such standard equipment. The 30-minute chromatography time provides for rapid turnaround for emergency diagnosis and monitoring of neonates with aminoacidopathies. The technic provides higher resolution than paper and thinlayer chromatography for separating the amino acids. The retention times are more reproducible and, hence, the interpretation and identification of amino acids is more objective. The rapidity of the analysis allows one to add standards to patient specimens for exact identification of peaks by coelution. Urine and serum use the same procedures. The separation of peaks is analogous to those seen on amino acid analyzers, and a new system of interpretation does not have to be learned. The system provides for semiquantitation, as well as quantitation for many amino acids. The use of the versatile HPLC equipment precludes the necessity for a hood or bulky highvoltage electrophoresis equipment and the expense of a dedicated amino acid analyzer. The development of our assay was self-restricted to use standard HPLC equipment, so that the analysis could be performed in any laboratory with HPLC gradient capabilities. Hence, most hospital laboratories can use this method as a screening tool and then refer samples for confirmation and definitive diagnoses. The advantages to the system exceed the few disadvantages. The system does require having HPLC equipment. The system is subject to interferences, although, even dedicated amino acid analyzers will separate and detect nihydrine-positive compounds that are not in the amino acid group. The derivative system used was chosen because of its rapid reaction time, stable reaction products, ease in reproducibility, and lack of requiring additional specific equipment. The dansyl chloride derivatization, however, forms multiple products with polyfunctional amino acids, preventing reliable quantitation of these species. Nevertheless, elevations of these compounds are readily detected as broad amorphous peaks, in contrast to the sharp peaks seen with most of the amino acids. A variety of HPLC technics for amino acid analysis, mainly of protein hydrolyzates, have been reported. Each is subject to its own advantages and disadvantages. Postion-exchange column ninhydrine derivatization requires a heated reaction coil. Like dedicated amino acid analyzers, proline and hydroxyproline form complexes with different absorbance maximums from the other amino acids. Additional pumps and mixers are required for postcolumn o-phthalaldehyde (OPA) derivatization. 6 Precolumn OPA derivatives are rapidly degraded, 12 so that derivatization must take place at a fixed time before injection, in order to maintain relative peak heights, and secondary amino acids, e.g., cystine, are not well derivatized. A variety of precolumn dansyl chloride derivatization methods have been reported, 4 " 14 and our new method is an optimization of these investigations. We found that substitution of acetonitrile 4 for acetone 14 as a solvent for dansyl chloride eliminated several extraneous peaks resulting from dansyl-cl-acetone reaction products. Mild reaction con-

618 RUTLEDGE AND RUDY A.J.C.P. May 1987 ditions, room temperature 14 versus 60 C, 4 " resulted in less side reactions with polyfunctional amino acids, lending additional specificity to the chromatogram. Substitution of lithium carbonate 4 " for sodium carbonate 14 buffered to a ph of 8.5 14 versus 9.5 4 " allowed the reaction to proceed to completion within 30 minutes rather than four hours or overnight. 14 Precolumn derivatization with phenylisothiocyanate yields a derivative that has stability, lacks reagent interference, and reacts with even the secondary amino acids. 2 This derivative shows great promise, and its sensitivity allows amino acid quantitation from dried blood spot specimens. HPLC appears to be the evolving method of choice for, not only amino acid qualitative results, but quantitative amino acid analysis. Our system employs a relatively simple HPLC system. One would predict enhanced results from use of additional gradient programming features and multiple wavelength detection or even diode array detectors to ensure that the derivatized peak corresponds to the amino acid having a particular retention time. While our technic does not require such equipment, some laboratories may desire to adapt the analysis to more versatile equipment. Using this system for more than one year, we have found that it performs satisfactorily. It meets our expectations insofar as degree of expertise required and ongoing expenses. It has, in many situations, provided information for reaching a preliminary diagnosis in the early initiation of therapy before confirmation of amino acid derangements in a variety of inborn errors of metabolism. Rapid amino acid analysis in the laboratory will enable a broader range of hospitals to provide support for diseases and therapies that require sophisticated analysis. The rapidity with which the system can analyze specimens allows same-day turnaround time, therefore, allowing laboratory support of a variety of new therapeutic modalities. Acknowledgment. The authors thank Gail Troxell for secretarial assistance. References 1. Andersen GE, Bucher D, Friis-Hansen B, et al: Plasma amino acid concentrations in newborn infants during parenteral nutrition. JPEN 1983; 7:369-373 2. Bidlingmuyer BA, Cohen SA, Tarvin TL: Rapid analysis of amino acids using pre-column derivatization. J Chromatogr 1984; 336: 93-104 3. Bremer HJ, Duran M, Kamerlin JP, et al: Disturbances of amino acid metabolism: Clinical chemistry and diagnosis. Baltimore, Urban and Schwarzenberg, 1981 4. De Jong C, Hughes GJ, van Wieringen K, et al: Amino acid analysis by high performance liquid chromatography: An evaluation of the usefulness of pre-column Dns derivatization. J Chromatogr 1982;241:345-359 5. Freund H, Dienstag J, Lehrich J, et al: Infusion of branch chainenriched amino acid solution in patients with hepatic encephalopathy. Ann Surg 1982; 196:209-220 6. Ishida Y, Fujita T, Asai K.: New detection and separation method for amino acids by high performance liquid chromatography. J Chromatogr 1981; 204:143-148 7. Joseph F Jr, Thurmon TF: Amino acid screening an objective approach. Laboratory Medicine 1983; 14:427-430 8. McCullough AJ, Czaja AJ, Jones JD, et al: The nature and prognostic significance of serial amino acid determinations in severe, chronic, active liver disease. Gastroenterology 1981; 81:645-652 9. Nyhan WL: Abnormalities in amino acid metabolism in clinical medicine. Norwalk, Appleton-Century-Crofts, 1984 10. Silverman LM, Christenson RH, Grant GH: Amino acids and proteins, Textbook of clinical chemistry. Edited by W Tietz. Philadelphia, WB Saunders, 1986, p 545 11. Tapuhi Y, Schmidt DE, Linder W, et al: Dansylation of amino acids for high performance liquid chromatography analysis. Anal Biochem 1981; 115:123-129 12. Turnell DC, Cooper JDH: Rapid assay for amino acids in serum or urine pre-column derivatization and reversed phase liquid chromatography. Clin Chem 1982; 28:527-531 13. Voelter W, Zech K: High performance of liquid chromatographic analysis of amino acids and peptide hormone hydrolyzates in the pico mole range. J Chromatog 1975; 112:643-649 14. Wiedmeier VT, Porterfield SP, Hendrick CE: Quantitation of Dnsamino acids from body tissues andfluidsusing high performance liquid chromatography. J Chromatogr 1982; 231:410-417