Automated pre-column derivatization and its application to amino-acid analysis using high-performance liquid chromatography

Although chemical modification of analytes is a well-accepted adjunct to chromatography, little attention had been paid to the automation of pre-column derivatization techniques. An automated pre-column system would offer advantages when analyses are performed using labile derivatives; and also when the time between injections is long and so staff time spent on achieving maximum analytical output is less than optimal. Both of these features exist with the analysis of amino-acids in physiological samples using pre-column 0-phthalaldehyde! 2-mercaptoethanol (OPA/MCE) derivatization and reversedphase high-performance liquid chromatography (HPLC) [1]. These fluorescent derivatives are labile !-1 and 2-1 and therefore have to be prepared in a precise manner immediately before injection onto the column. A system is described for automatically sampling, derivatizing and injecting specimens onto an HPLC and analysing for amino-acids. Furthermore, this system may be operated overnightmcapitalizing on the reduced chromatographic run time when compared with amino-acid analysis using ionexchange post-column derivatization methods [3].


Introduction
Although chemical modification of analytes is a well-accepted adjunct to chromatography, little attention had been paid to the automation of pre-column derivatization techniques. An automated pre-column system would offer advantages when analyses are performed using labile derivatives; and also when the time between injections is long and so staff time spent on achieving maximum analytical output is less than optimal.
Both of these features exist with the analysis of amino-acids in physiological samples using pre-column 0-phthalaldehyde! 2-mercaptoethanol (OPA/MCE) derivatization and reversedphase high-performance liquid chromatography (HPLC) [1]. These fluorescent derivatives are labile !-1 and 2-1 and therefore have to be prepared in a precise manner immediately before injection onto the column.
A system is described for automatically sampling, derivatizing and injecting specimens onto an HPLC and analysing for amino-acids. Furthermore, this system may be operated overnightmcapitalizing on the reduced chromatographic run time when compared with amino-acid analysis using ionexchange post-column derivatization methods [3].

Materials and methods
Instrumentation The sample derivatization system comprised a Magnus MT100 sampler fitted with a pneumatically-operated Rheodyne 7010 ;alve (Magnus Scientific Ltd, Aylesbury, UK), a Technicon AA1 pump fitted with a 0.32ml/min and a 0"10ml/min pump tube and associated autoanalyser components (Technicon Instruments Company Ltd, Basingstoke, UK). Chromatographic separations were performed using an Altex 420 gradient HPLC with a 150 x 4"6 mm i. d. column pre-packed with 5/m diameter Ultrasphere ODS (Anachem Ltd, Luton, UK) and a Schoeffel FS970 fluorescence detector (Kratos, Manchester, UK). Chromatographic data was processed by a SP4100 computing integrator (Spectra-Physics Ltd, St. Albans, UK).

Modifications to M7100 sampler
The original minipump supplied with the sampler was disconnected. The 240 VAC power-supply to this pump was fed to a 240 VAC-110 VAC step-down transformer and, in turn, to the Technicon AA1 pump. The 110 VAC supply line was also fed to a two-pole change-over octal relay (115 VAC), which controlled the supply to the pneumatics of the Rheodyne valve. This enabled simultaneous switching of the injection loop and control of the Technicon AA1 pump. 36 Double-lumen probe Both of the axial nipples of a PT 9 connector (Technicon Instruments Company Ltd) were removed. One was replaced with a 3 cm length of a stainless-steel needle (0.8 mm i.d.  o.d.). A stainless-steel capillary tube, 9cm long (0.4mm i.d. 0"6 mm o.d.), replaced the other axial nipple so that one end was approximately mm above the needle tip (see figure 1).
The junction between the capillary tube and the top of the connector was filled with epoxy cement. By using a larger flow rate at A (figure 1) than at B, the sample is drawn into the probe at C at a rate equal to the difference between the flow rate at A and B. The probe is connected to the injection valve with Teflon tubing (0.3 mm i.d. 1"5 mm o.d. OPA/MCE reagent: dissolve 500mg of OPA (Sepramar grade) in 10ml of methanol and dilute to 100ml with. a 400mmol/1 sodium borate buffer solution (pH 9.5). To this solution add 400/A of 2-mercaptoethanol followed by a further 40 #1 every three days. When stored in a brown-glass bottle this reagent was stable indefinitely at room temperature. Iodoacetic acid reagent: dissolve 0.74 g ofiodoacetic acid and 0.62 g of boric acid in approximately 50 ml of water. Adjust the pH to 9"5 with 2 mol/l sodium hydroxide solution and dilute to 100ml with water. This reagent is stable indefinitely at room temperature. Chromatographic conditions and quantitation The chromatographic conditions used have been previously described [1]. Amino-acid derivatives were identified by their relative retention times and quantified by comparing their peak areas with that of homocysteic acid and the internal standard.

Stability of samples treated with iodoacetic acid
A pooled human serum specimen was prepared for analysis using the sample treatment procedure described. A 40/A aliquot of this sample was added to 20 pl of OPA/MCE reagent in a polypropylene tube. The contents of the tube were mixed, and 201 immediately injected onto the column using a 7125 Rheodyne 20 pl loop injection valve (from Anachem Ltd). This manual derivatization and injection procedure was repeated on the same sample every 2 h for 24 h. The stability of a standard and urine specimen were investigated in the same manner. Over the period, no significant variation was observed in the concentrations of the amino-acids.

Derivatization precision
The performance of the derivatization system was examined by estimating the within-run assay variance, with and without reference to the internal standard (see table 1). The amino-acids quantified were selected to provide retention times ranging throughout the chromatogram.
These precisions represent the reproducibility of (a) the volume of sample aspirated; (b) the mixing of the sample with OPA/MCE reagent; (c) the yield of the derivatives; (d) the time delay between sampling and loading; and (e) the filling of the injection valve loop.
Sample carry-over A 1.0mmol/1 standard amino-acid solution was prepared for sampling. Using a cycle time of 5 min (to ensure that the system was completely filled with derivatized sample), the standard solution was assayed without the presence of internal standard. Immediately afterwards, a water sample was aspirated with a cycle time of 15 s and the amino-acids quantified again. This procedure was repeated increasing the cycle times of the water sample (see table 2). The cycle time for the system was set at 60 s when negligible carry-over occurred.

Discussion
Ideally, derivatization of analytes before or after chromatographic separation should be avoided because it imposes further potential errors in techniques. If, however, for such reasons as D. C. Turnell and J. D. H. Cooper Automated pre-column derivatization detection, specificity or chromatographic resolution, it becomes necessary to derivatize analytes, the method used should be one with minimal adverse effect on the performance ofthe analytical technique. Pre-column derivatization is superior to postcolumn derivatization in that its effect on analytical performance is minimal for the following reasons: (a) it does not involve an increase in path-length between the column and detector and therefore does not decrease separation efficiency; and (b) postcolumn derivatization equipment has to be engineered to operate continuously within narrow tolerance limits because internal standards cannot be incorporated to correct for all possible variations in performance. The design of the pre-column derivatization system had to fulfil two basic requirements; firstly, the ratio of the amount of sample loaded to the amount of sample consumed had to be large; and, secondly, the continuous-flow system had to operate discontinuously with a short cycle time in order to conserve reagents.
The double-lumen probe permits samples to be aspirated, well mixed with reagents and transferred directly to the injection valve. This minimizes the volume of tubing that the sample passes through and thus reduces the volume of sample required to purge the system of the previous specimen.
In conventional continuous-flow systems, flow rates take some time to stabilize due to the compressibility of the air segmentation used to assist mixing and minimize sample diffusion. If air segmentation was to be used in this application, the air would have to be removed before the injection and would grossly extend the sampling time. The unsegmented stream used in this system permits rapid stabilization offlow rates, with a low volume and simple peristaltic tubing configuration.
Because the double-lumen probe is connected to both the inlet and the outlet of the peristaltic pump, the inherent pulsing observed with this instrument is amplified at the probe inlet (see C in figure 1). To reduce this effect, it is necessary that the flow rate ratio A/B (figure 1) be no less than 3. Also, to obtain acceptable precision the injection valve has to be turned from load to inject while the sample stream is flowing through the loop (table 1). This was achieved in this system with the sampler modification.
The precision of the system in the absence of the internal standard (table 1) demonstrates that stable flow rates exist and that, despite the absence of air segmentation, the mixing of the sample and reagent is adequately reproducible and carry-over is minimal. Moreover, the larger coefficients of variation obtained when the internal standard was used for the quantitation indicates that the errors of chormatography and integration were greater than the errors of sampling and derivatization. The double-lumen probe further reduces the time taken for the sample to pass to the injection loop by introducing it immediately into the sample/reagent stream. Even when derivatization is not required, this feature ofthe double-lumen probe is useful for rapidly loading samples automatically onto a liquid chromatograph.
When the double-lumen probe is used in conjunction with an unsegmented-flow technique, a minimum amount of sample may be derivatized and loaded in the shortest possible cycle time in a discontinuous mode.