Journal of Automatic Chemistry of Clinical Laboratory Automation, Vol. 7, No. 4 (October-December 1985), pp. 177-180

Automated preparation of biological samplesprior to high pressure liquidchromatography: Part I- The use of dialysis

deproteinizing serum for amino-acidanalysis

D. C. Turnell and J. D. H. CooperDepartment ofBiochemistry, Coventry and Warwickshire Hospital, Stoney StantonRoad, Coventry CV1 4FH, UK

Introduction

All methods for the analysis of amino-acids in serum bycolumn liquid chromatography require initial deproteini-zation of the sample. This is typically performed usingsulphosalicylic or perchloric acids for ion-exchangechromatography ], or acetonitrile when using reversedphase chromatography [2]. However, these techniquesare difficult to automate without the use of sophisticatedrobots.

Although deproteinization using dialysis should not, inprinciple, suffer from these disadvantages, the resultingdialysate would contain sub-analysable concentrations ofamino-acids. However, the high sensitivity ofpre-columno-phthalaldehyde/2-mercaptoethanol (OPA/MCE) deri-vatization and reversed phase high pressure liquidchromatography (HPLC) methods [2-4] permitted theexamination of the potential of this deproteinizationtechnique.

This paper describes the use ofdialysis for the deproteini-zation of serum samples for amino-acid analysis and itsincorporation into an automated pre-column derivatiza-tion HPLC system [5].

Materials and methods

Instrumentation

The gradient HPLC used comprised an Altex 420 system,a 150 x 4.6 mm column prepacked with 5 tm Ultra-sphere ODS (Beckman-RIIC Ltd, High Wycombe, UK)and a Schoeffel FS970 fluorescence detector (Kratos,Manchester, UK) using an excitation wavelength of 240nm and an emission cut-off filter of 417 nm. Sampleinjection was performed with a pneumatically operatedRheodyne 7010 valve (Anachem Ltd, Luton, UK) fittedwith a 50 il loop. Chromatographic data was processedby a SP4100 computing integrator (Spectra-Physics Ltd,St Albans, UK). The gradient program and HPLCmethod have been previously described [2].

Dialysis and derivatization system

The derivatization system (figure 1) comprised a modi-fied auto-analyser (AA) sampler II, an AAI pump and a6 in AAII dialyser block fitted with a C-type cuprophanemembrane, which has a molecular weight cut-off of 1000(Technicon, Basingstoke, UK). The sampler was fittedwith a double lumen probe that was constructed aspreviously described [5]. The autosampler was modifiedby removing the timing cam and motor accessory andreplacing the associated microswitch with a miniatureopen single pole change-over 5 V relay (Radio Spares,London) connected to one of the six external eventoutputs (T2-T8) of the SP4100. The power supply to theinjection valve actuator and the AA1 pump were alsocontrolled by the SP4100 external event outputs viaminiature relays. All the relays were fitted with clampingdiodes to avoid transistor overload. A BASIC program inthe integrator refers to the run time clock to switch theexternal event outputs during the process cycle (figure 2),starts the Altex 420 gradient programmer followinginjection and halts the system after the required numberof samples have been analysed.

Reagents

All amino-acids, iodoacetic acid and 2-mercaptoethanolwere obtained from Sigma London Chemical Company

() WasteDialyser

- Probe HPLC Pumps’ HPLC ColumnSampleLoop

Figure 1. Schematic diagram ofthe automated system. Flow rates(ml/min) ofpump tubes 1 0"16, 2 0"8, 3 0"16.

177

D. C. Turnell and J. I). H. Cooper Automated sample preparation for HPLC: Part

Loadam___ple Dialysis valve,

InProbe

Out

Wash system

Chromatography and integration

InjectInjectionvalve

Load

10

4

8

18 23

12

19

15 2

13

0 100 200 300Time (secs)

Figure 2. Timing diagram ofthe operational sequence ofevents inthe automated system.

Ltd, Poole, UK. Acetonitrile Far UV grade was obtainedfrom Fisons Scientific Apparatus, Loughborough, UK.Unless otherwise stated, all other chemicals were analy-tical grade and were supplied by BDH Chemicals Ltd,Poole, UK. Water purified through activated carbon andan ion-exchange resin (Spectrum C system, Elga Ltd,High Wycombe, UK) was used for all reagent prepara-tions.

OPA/MCE

100 mg of OPA was dissolved in l0 ml of methanol anddiluted 100 ml with 200 mmol/1 sodium borate buffersolution (pH 9.5). To this was added 200 1 ofMCE andthe reagent stored in a brown glass bottle.

lodoacetic acid

5.0 g of iodoacetic acid was dissolved in approximately80 ml of sodium borate buffer solution (200 mmol/l, pH9.5) and the pH adjusted to 9.5 with 4 mol/1 sodiumhydroxide solution.

Statdard amino-acid solutionThe amino-acid standard was an ac_lueous 500 tmol/1solution of each amino-acid listed in figure 3.

Internal standard solution

The stock internal standard was an aqueous 10 mmol/1solution of homocysteic acid, homoserine and norvaline.Aliquots of both the standard and internal standardsolutions were stored at -20C. Working internalstandard solution was prepared by diluting ml of thestock solution to 50 ml with water; 200 1 of MCE wasadded to this.

Procedures

Sample preparationFor routine procedures, 50 tl of serum was added to 500tl of working internal standard solution and placed in a

32

202

4

18 23 0

11

1214 16

10

Figure 3. Chromatograms of (a) a serum from a one-month-oldchild suffering from hyperphenylalanaemia. (b) An aqueousstandard containing 500 lamol/l of each amino-acid. Both chro-matographs were obtained using the automated dialysis procedure.The amino-acids are identified as follows: 1, phosphoserine; 2,aspartic acid; 3, glutamic acid; 4, cystine derivative; 5,o:-aminoadipic acid; 6, asparagine; 7, homocystine derivative; 8,serine; 9, histidine; 10, glutamine; 11, ethanolamine phosphoricacid; 12, glycine; 13, threonine; 14, citrulline; 15, arginine; 16,3-methylhistidine; 17, [3-alanine; 18, alanine; 19, taurine; 20,tyrosine; 21, o-amino-n-butyric acid; 22, ethanolamine; 23,valine; 24, methionine; 25 tryptophan; 26, L-cystathionine; 27,phenylalanine; 28,L-isoleucine; 29, leucine; 30, hydroxylysine;31, ornithine; 32, lysine. ISI homocysteic acid, IS2homoserine, IS3 norvaline.

sample cup on the auto-sampler. MCE in the internalstandard solution reduces cystine and homocystine tocysteine and homocysteine respectively.

Derivatization systemThe configuration of the dialysis and derivatizationsystem is shown in figure 1. The timing diagram for atypical cycle for treating one sample is shown in figure 2and operates as follows: the integrator initiates thesampler to introduce the double lumen probe into thesample. Sample is aspirated together with iodoaceticacid, which acetylates sulphydryl containing amino-acids[6]. After aspiration of the sample the pump is switchedoff, the probe is returned to the wash on the sampler andthe amino-acids are allowed to diffuse across the dialysermembrane into the OPA/MCE reagent. One minute

178

D. C. Turnell and J. D. H. Cooper Automated sample preparation for HPLC’ Part

later the OPA/MCE pump is energized and the amino-acid OPA/MCE derivatives are moved into the injectionloop. The injection loop is then switched from load toinject and the derivatized amino-acids eluted onto thecolumn. After 10 s the injection valve is switched to load,and the residual derivatives and sample are purged fromthe system. The system then shuts down to await the endof the chromatography and integration report.

Chromatographic conditions and quantitation

The chromatographic conditions used have been des-cribed previously [2]. Amino-acid derivatives were iden-tified by their relative retention times and quantified bycomparing their peak areas with that for homocysteicacid (internal standard).

System design and validation

Process times

The times of events in the process cycle depend on thefollowing: (1) dialyser size or volume; (2) volumes ofconnecting tubing; (3) pump tube flow rates; (4) size ofinjection loop; and (5) analytical recovery of analytes.

The volume ofthe donor and recipient channels ofthe 6 indialyser block was 120 1 and 0.8 mm I.D. teflon tubingwas used for all connections. With a 50 1 injection valveloop, and pump tubes of the size indicated in figure 3, thetravel of a sample slug through the system was assessedby introducing a small air segment into the sample andrecipient sides of the system with the pumps energizedand timing their progress. The time for an air segment totravel from the tip of the sample probe to the inlet (donorside) of the dialyser was 6 s, from the inlet to the outlet ofthe dialyser it was 13 s, and from the outlet ofthe dialyser(recipient side) to the outlet of the injection loop the timewas 30 s. From these figures, it is possible to determinethe time required to completely fill the dialyser and themoment at which the Rheodyne valve should be switchedfrom load to inject. The effect ofvarying the dialysis timeon the areas of the chromatographed derivatives ofhomocysteic acid and serine is shown in figure 4. Serinewas used due to the high stability of its OPA/MCEderivative [7].

1o 2bo 3bo 4bo sbo’ 8bo 7bb 860Dialysis (seconds)

Figure 4. The effect ofdialysis time on the recovery ofhomocysteicacid & A and serine Z /k.

Precision

The within- and between-run assay variances of theanalysis of serine, threonine, tyrosine and methionine inboth an aqueous standard and pooled serum are shown intable 1.

AccuracyThe mean analytical recovery was 101% (SD 2"3%), asestimated from analysis of samples supplemented withknown quantities ofamino-acids. Analytical linearity wasonly lost if the fluorimeter response exceeded 1"5 timesfull-scale deflection. However, since quantitation is basedon peakarea and not peak height, the linearity maximafor each amino-acid depends on both the peak shape andthe specific fluorescence intensity of the OPA/MCEderivative. The method was linear with concentration upto a minimum of 1800 mol/1.

Matrix interferenceThe magnitude ofmatrix interference was determined byinvestigating the recovery of homocysteic acid (notusually present in normal serum) from specimens withvarious concentrations of serum. Serial dilutions of apooled serum with water were supplemented with thesame volume of homocysteic acid solution (1.0 retool/l).An aliquot of the same pooled serum was supplementedwith the homocysteic acid solution and then seriallydiluted with water. These samples were loaded directlyonto the autosampler omitting pre-dilution with internalstandard solution and the areas of the homocysteic acidpeaks estimated. The peak areas obtained from analyzingthese samples are shown in figure 5. The linear responsesobtained indicate that the concentration of serum doesnot affect the recovery of homocysteic acid. With theexception of tryptophan, all the amino-acids listed infigure 3, showed identical response relationships. Withserum dilutions of less than one in five, tryptophanexhibited a reduced recovery.

Dialysis efficiency

The peak area resulting from known amounts of amino-acids on column was determined by manually derivatiz-ing an aqueous standard solution and injecting onto theHPLC using a filled loop technique (20 btl) with aRheodyne 7125 valve. The same stafidard solution was

Table 1. Precision ofassaying a standard solution (500 ltmol/1)and a normal serum.

Within-run Between runN 20 N 20CV% CV%

Aqueousstandard

Serum

Serine 1"8 2’6Threonine 1"9 2"9

Tyrosine 1"5 3"5Methionine 1.1 2"4

Serine 1.3 1"9Threonine 2"5 3"5Tyrosine 2"2 3"6Methionine 3"8 5"5

179

D. C. Turnell and J. D. H. Cooper Automated sample preparation for HPLC: Part

Figure 5. Peak areas obtained from (a) a diluted serumsupplemented with the same amount ofhomocysteic acid/h---/h; (b) & i dilutions of a serum that was initiallysupplemented with homocysteic acid.

then dialysed and derivatized using the automatedsystem and the absolute peak areas determined. Fromthese areas it was calculated that 13% of the amino-acidsin the sample had passed across the dialyser membraneand derivatized.

Discussion

Although the pre-column derivatization of amino-acidshas been successfully automated [5], the samples stillrequired initial manual deproteinization. The combina-tion of dialysis with the derivatization system providesone way to totally automate sample preparation foramino-acid analysis.

The selected size of the dialyser is dependent on thesensistivity required. Although the use of a large dialyserdoes not increase the recovery of amino-acids from thesample, ifone is used in conjunction with a large injectionloop, the amount of amino-acid derivatives loaded ontothe HPLC is increased. For precise injection of deriva-tives, it is essential to use an injection loop with a smallervolume than that of the dialyser channel.

The 60 s dialysis time used does not allow maximumrecovery ofanalytes but yields an adequate sensitivity in arelatively short time. Although at 60 s the recovery ofamino-acids is still increasing (see figure 4), the system isquantitatively precise (table 1) due to reproducibleprocess timing via the SP4100 integrator. The effect ofserum concentration on the recovery of tryptophan isconsistent with protein binding of this amino-acid whichhas been previously reported [8].

Where relatively large volumes of sample are available,and maximum sensitivity is not required, the process maybe simplified by eliminating the stopped flow step,allowing the amino-acids to dialyse and derivatize in acontinuous flow mode. Using a smaller OPA/MCE flowrate (0.16 ml/min) than the sample/IDA flow rate (0.8ml/min) serves to reduce the dilution ofderivatives in thedialysate. Counter current dialysis did not improve theobserved sensitivity.

The exact process times adopted depend on the flow ratesof the pump tubes and volumes of the connecting tubes

180

used. Precise times therefore would need to be establishedempirically for each individual system.

A major consideration with the design of a sampler forHPLC is the proportion of sample loaded compared withthe minimum volume required to be in the sample cup.Ideally, samplers should be able to inject the entirecontents of the sample cup, but in practice this isimpossible. When the sampler is also required to performderivatization, a further problem is caused by the smallvolume of the injection loop relative to the dead volumebetween the sample cup and the outlet of the injectionloop. In this system the load volume: dead volume ratio ismaximized by using a double lumen probe and stoppedflow dialysis. Using the do.uble lumen probe, sampleentering the system is immediately mixed with IDAreagent. The volume of sample aspirated has to com-pletely purge and fill the donor channel of the dialyser,and is dependent on both the size of the dialyser used andthe dead volume between the probe and the dialyser inlet.With this system, approximately 37% of the sampleaspirated is loaded into the dialyser donor channel, 13%of the amino-acids pass into the recipient channel and42% of the dialyser channel volume is loaded into theinjection valve loop. Thus the equivalent of 2% of theamino-acids sampled is injected onto the HLPC column.However, as the HPLC method has a sensitivity of 38fmoles on column of each amino-acid [2], samplescontaining 6 nm01/1 of amino-acids may be analysed bythe system. The duration of the wash sequence was notoptimized with respect to reagent consumption but wasadequate to eliminate carry-over. Although excessreagent is consumed, it is insignificant when comparedwith post-column derivatization where reagent ispumped continually during the chromatography. Fur-thermore, as the wash sequence occurs while the analytesare being chromatographed, its duration does not influ-ence the time for one total analytical cycle.

By combining automatic dialysis and derivatization, theanalysis ofamino-acids in complex matrices is facilitated.The system performs sampling, dialysis, derivatizationwith IDA and OPA/MCE, together with injection ontothe HPLC, in only 135 s. Furthermore the efficiency ofthis clean-up procedure prolongs the life of the analyticalHPLC column without employing guard columns.

References

1. MOORE, S., SPACKMAN, D. H. and STEIN, W. H., AnalyticalChemistry, 30 (1958), 1185.

2. TURNELL, D. C. and COOPER,J. D. H., Clinical Chemistry, 28(1982), 527.

3. GRIFFIN, M., PRICE, S. J. and PALMER, T., Clinica ChimicaActa, 125 (1982), 89.

4. HOOAN, D. L., KRAEMER, K. L. and ISENBERO, J. I.,Analytical Biochemistry, 127 (1982), 17.

5. TURNELL, D. C. and COOPER, J. D. H., Journal ofAutomaticChemistry, 5 (1983), 36.

6 COOPER, J. D. H. and TVRNELL, D. C., Journal of Chroma-tography, 227 (1982), 158.

7. COOPER, J. D. H., OODEN, G., MCINTOSH, J. and TURNELL,D. C., Analytical Biochemistry, 142 (1984), 98.

8 MCMENAMY, R. H., LUND, C. C., VAN MARCKE, J. andONC.EY, J. L., Arch. Biochem. Biophys., 93 (1961), 135.

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