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Aug 23, 2006 - ANALYSIS IN TWO TISSUES BY PITC- AND OPA-DERIVATIZATIONS, Analytical Letters, 34:6,. 867-882, DOI: 10.1081/AL-100103598.
Analytical Letters

ISSN: 0003-2719 (Print) 1532-236X (Online) Journal homepage: http://www.tandfonline.com/loi/lanl20

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY INTERCOMPARATIVE STUDY FOR AMINO ACID ANALYSIS IN TWO TISSUES BY PITC- AND OPADERIVATIZATIONS Pasquale Avino , Luigi Campanella & Mario Vincenzo Russo To cite this article: Pasquale Avino , Luigi Campanella & Mario Vincenzo Russo (2001) HIGHPERFORMANCE LIQUID CHROMATOGRAPHY INTERCOMPARATIVE STUDY FOR AMINO ACID ANALYSIS IN TWO TISSUES BY PITC- AND OPA-DERIVATIZATIONS, Analytical Letters, 34:6, 867-882, DOI: 10.1081/AL-100103598 To link to this article: http://dx.doi.org/10.1081/AL-100103598

Published online: 23 Aug 2006.

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Date: 20 September 2015, At: 22:05

ANALYTICAL LETTERS, 34(6), 867–882 (2001)

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BIOANALYTICAL

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY INTERCOMPARATIVE STUDY FOR AMINO ACID ANALYSIS IN TWO TISSUES BY PITC- AND OPA-DERIVATIZATIONS Pasquale Avino,1,* Luigi Campanella,1 and Mario Vincenzo Russo2 1

Dipartimento di Chimica, Universita´ di Roma ‘‘La Sapienza,’’ piazzale Aldo Moro 5, 00185, Roma, Italy 2 Facolta´ di Agraria (DiSTAAM), Universita´ del Molise, via De Sanctis, 86100, Campobasso (CB), Italy

ABSTRACT In this paper, we have extensively investigated the pre-column derivatization method with o-phthaldialdehyde (OPA) to analyze cysteine, cystine, among with other 18 amino acids, using reversed-phase high-performance liquid chromatography analysis (RP-HPLC), and after we have compared it with an other method involving phenylisothiocyanate (PITC) in order to have a two sensitive methods to use accordingly the matrix. We have studied the analytical conditions for the OPA-reaction together with precision, linearity range, correlation coefficient (>0.998) and

* Corresponding author. Current address: DIPIA-ISPESL, via Urbana 167, 00184, Rome, Italy. E-mail:[email protected] 867 Copyright & 2001 by Marcel Dekker, Inc.

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limit of detection (between 14 fmol and 1 pmol) for each amino acid. Then, we have applied the two methods to the analysis of larynx and kidney tissues: between the two methods, the amino acid recoveries (between 80% and 100%) and the correspondence of the OPA and PTC results are satisfactory considering the different involved derivatization procedures. PTC- and OPA-chromatograms obtained in the analysis of actual samples are reported together with the amino acid level, with particular regard to the cysteine/cystine determination. Key Words: Amino acids; Comparison; Cysteine; Phenylisothiocyanate; o-Phthaldialdehyde.

INTRODUCTION In clinical chemistry the amino acid (AA) analysis in biological fluids and tissue is very important for checking and diagnosing metabolic diseases. Until now, because of physiopathological and practical reasons the AAs have been widely studied in plasma and urine [1–6]. In fact, e.g., the AA analysis of biological fluids from patients affected by hepatic pathologies has exhibited different amounts of cystine, taurine, methilhistidine and methionine according to the state of progress of the pathology [7]. Basically, the evaluation of the AA alteration from the normal values is a powerful tool against the advancement of some disorder. Nowadays, a large interest is focused on the interaction between AA and toxic pollutants in human target organs: recent theories hypothesize that some heavy metals (e.g., Cd and Pb) are able to join to proteic sulfur group (-SH) to inhibit enzymatic activity [8]. In particular, some kinds of cancer (e.g., thyroidal, laryngeal, and kidneal cancers) are speculated to be related to anomalous cysteine levels. In the AA analysis, the reversed-phase high-performance liquid chromatography (RP-HPLC) is the commonest analytical technique used. In a previous paper [6], we have extensively described an extraction scheme and an analytical procedure based on derivatization with phenylisothiocyanate (PITC) to determinate 20 AA, including cysteine and cystine in biological fluids and tissue. In this paper we concentrate the attention on an other pre-column derivatization method using o-phthaldialdehyde (OPA): this reagent reacts quickly with primary AAs to form highly fluorescent derivatives [9,10]. The method is based on the reaction between the amino group of AA

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and the OPA when a thiolic reagent and high pH are present [11,12]. Many modifications have followed the first protocol but many problems are still ‘‘opened’’. For example, the optimal conditions for the reaction between AAs and OPA are not so clear in spite of the wide literature [13–16]. The amount of 2- mercaptoethanol (MCE) [10,14], the pH of the reaction mixture and the reaction time [10,17] are only the more common problems studied by many authors but are also crucial for the optimization of the AA analysis. Each of these tasks is relevant to optimize the reaction: i) the MCE amount controls the OPA-complex stability, ii) an optimal pH of the reaction mixture avoids a rapid decrease of fluorescence, and iii) the reaction time allows the OPA-AA reaction to be completed. In 1996 Dorresteijn et al. [10] published a paper where the optimal conditions using OPA with MCE were investigated: they found a limit of detection (0.05–0.43 pmol) and reproducibility (0.2–2.2 %) for all primary AAs except cysteine, using MCE concentration of 1.6 mg/mL, reaction time of 1 min. Other problems are connected with significant decay in fluorescence of glycine derivative, poor response of OPA-lysine, lack of reaction of OPA with the AAs proline and hydroxyproline [9]. Finally, the simultaneous determination of cystine and cysteine is not a trivial task. To overcome this drawback, cysteine and cystine are treated with iodioacetic acid (IA) to obtain a S-carboxymethilcysteine (CMC) derivative [17], which reacts with OPA to yield a fluorescent derivative. In this paper we find the optimal conditions for the OPA reaction. Special regards are dedicated to explicate the method (i.e., linearity range, correlation coefficient, limit of detection (LOD) of each AA) and to emphasize the simultaneous determination of cysteine, cystine and other 18 AAs with the OPA method. After, we compare the applicability and the results obtained in the analysis of two human tissues (kidney and larynx) with the two pre-column derivatization RP-HPLC methods (OPA and PITC) to have cross-checking data for compounds of such difficult determination, especially when a chemical pre-treatment is required. This is an important task of this work; in fact, the reproducibility of both the methods, especially for the OPA-method, is not easy because many papers [19] underlines the no good reproducibility of this method. EXPERIMENTAL 2.1

Solvents and Reagents

A Milli-Q water purification system (Millipore, Bedford, MA, USA) was used to prepare mobile phases, standard solutions and buffers. HPLC grade acetonitrile and methanol were used while anhydrous sodium acetate,

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dipotassium hydrogenphosphate, sodium dihydrogenphosphate, ethanol and acetic acid were all analytical grade from Carlo Erba (Milan, Italy). HCl 6 N, triethylamine (TEA), and phenylisothiocianate (PITC), all ‘‘sequanal grade’’, and ‘‘amino acid standard H’’ containing arginine (Arg), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), metionine (Met), phenylalanine (Phe), tyrosine (Tyr), threonine (Thr), valine (Val), alanine (Ala), aspartic acid (Asp), glitamic acid (Glu), glycine (Gly), proline (Pro), serine (Ser) and cystine ((Cys)2) (2.5 mmol each ones except (Cys)2, 1.25 mmol) were purchased from Pierce (Rockford, IL, USA). Individual amino acids (‘‘Kit D-L Amino acids’’), included cysteine (Cys), homocysteic acid (Omo), S-carboxymethylcysteine (S-CMC), sulphosalicylic acid (SSA), OPA, 2-MCE and IA were purchased from Sigma (St. Louis, MO, USA).

2.2 2.2.1

Extraction and Derivatization Procedures

Preparation of Standard Solutions

For PTC-AA identification, the procedure involved is reported in a previous paper [6]. For qualitative and quantitative OPA-amino acid identifications, we used a standard solution obtained by mixing opportune volume of standard single amino acid solution (103). In this way, 5 to 200 mmol of each amino acid have been derivatized following the procedure described below.

2.2.2

Preparation of OPA Derivatizating Solution

The OPA derivatizing solution was prepared daily adding 9.6 mL of boric acid (3% w/v in water and pH adjusted to 10 with KOH) to 400 mL of a solution obtained adding 50 mL of MCE to 50 mg of OPA in 1 mL of ethanol. After 10 min, 50 mL of methanol were added. The solution was mixed and flushed with nitrogen and stored in the dark at 5 C.

2.2.3

Preparation of Samples

The analytical extraction is similar to that used for other matrices [6]. Both the tissues (kidney and larynx) were supplied from ‘‘Policlinico Umberto I’’ of University of Rome and stored at 25 C, to avoid degradation of some AAs [1], until ready to use. All the samples were processed within 1 hr of collection. Seventy-milligrams of each tissue were

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deprotenized by adding 5 volumes of 3% SSA, vortexing and centrifuging at 3600  g for 20 min. The solution was filtered through 0.45 mm filter (Whatman, ME, England) and processed as described below.

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2.2.4

Drying and Derivatization

The derivatization procedure with PITC is well described in the previous paper [6]. For the OPA analysis the procedure is more easy and quick than the PTC analysis. To 25-mL volume of the solution obtained from the previous step a 50-mL aliquot of homocysteic acid (I.S.) 10–5 M, was added. The solution was dried at 35 C by rotary evaporator (Bu¨chi, Switzerland) for 10 min and 500 mL of mobile phase A were added. To 200-mL volume of this solution 25-mL of OPA-derivatizating solution described above, were added. After 3 min, the OPA-reaction was complete and an aliquot was injected into the chromatographic system for the sample analysis. For the cysteine analysis, 200 mL of sample were previously added to 25 mL of I.S. and 25 mL of 100 mM IA. After standing for 15 min, the solution was dried and derivatized with OPA.

2.3

Chromatographic Equipment

In [6] the experimental conditions and the chromatographic equipment involved in the PTC-analysis are described. The HPLC equipment involved in the OPA-analysis was a Kontron mod. 422 (Kontron, CA, USA), equipped with a fluorescence detector Perkin-Elmer mod. 650 (Norwalk, CT, USA) set at  ¼ 330 nm in excitation and  ¼ 440 nm in emission. No heater module was used to keep the column temperature constant. Separations were carried out on a LC-18 Hypersil, 150.46 cm i.d. column, 5 mm. A homemade C-18 guard column was used to protect the analytical column. The sample was introduced with a Rheodyne valve equipped a 20 mL external loop. In the first 2 min the flow was linearity increased from 0.2 mL/min to 1.0 mL/min. Then, the mobile phase flow-rate was kept at 1.0 mL/min for all the time. A binary gradient was employed where mobile phase A was phosphate buffer (20 mM at pH 6.7)-methanoltetrahydrophurane (98:1:1, v/v), mobile phase B was phosphate buffer (20 mM at pH 6.7)-methanol (35:65, v/v). The gradient is reported on the chromatogram (Fig. 1a). Ten min was required to re-equilibrate the initial column condition.

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RESULTS AND DISCUSSION The PTC method has been widely studied in a previous paper [6] where the chromatograms and the data for each AA are reported. In this paper the authors wish to compare the PITC derivatization with the OPA derivatization. This second derivatization method is considered more sensitive between those based on fluorescence detection. Among the different thiolic agents, we have used 2-MCE without having relevant problems. The involved derivatization procedures are based on the following reactions (a for PTC- and b for OPA-analysis):

While the PITC method is clear enough, the OPA method is still in progress because the optimal conditions, reaction time, pH of the reaction mixture and the used solvent, are not investigated to have a protocol. At room temperature the 1-min OPA-reaction time is sufficient to have the maximum fluorescence but for our technical problems we have established a 3-min reactione time. In fact, experiments in this way confirm the fluorescence stability, instead after 25–30 min a fluorescence decay yield till 75%. The pH of the reaction mixture and the concentration of 2-MCE are the other important parameters of the OPA reaction. All these conditions allow the reaction of OPA with amino acids to complete with the maximum fluorescence. Finally, using these conditions there is no presence of interfering peaks, except in the cysteine analysis. In the previous paper we have demonstrated how the cysteine can be determined as PTC-S-carboxymethylcysteine together with the other PTCamino acids when there is a high level of AAs ( 25 nmol derivatized). Otherwise, when the AA levels are low (