Gas Chromatography of Plasma Amino Acids Using a Wide Bore Column: Comparison with Packed and Capillary Columns D. Labadarios*, 1. M. Moodie, J. A. Burger, and G. S. Shephard MRC Metabolic Research Group, Tygerberg Hospital, Department of Internal Medicine, University of Stellenbosch, P.O. Box 63, Tygerberg 7505, South Africa

Key Words:

Gas chromatography Fused-silica capillary Wide bore glass capillary Packed column Plasma amino acids

Summary

A procedure is described in which a wide bore glass capillary column is used as an alternative to the more traditional packed column in the analysis of amino acid levels in plasma. The coefficientsof variationforallaminoacids (with theexceptionof aspartic acid) were better than 1 1 O h with recoveries ranging from 81% to 122OI0. The data are compared with the corresponding results obtained using a packed column and show significant differences (p < 0.005) between values for glycine, serine, isoleucine, proline, methionine, aspartic acid, phenylalanine, and lysine. A similar comparison between resultsfrom the wide bore and the fused-silica open tubular (FSOT) column shows better agreement. Adjustment of chromatographic conditions for the wide bore analysis yields results in good agreement with those from FSOT analysis but which still differ significantly from the packed column data.

1 Introduction The use of gas chromatography (GC) in the determination of amino acids continues to become more widespread as judged by the appearance of increasing numbers of reports on the subject in the literature. Considerable success has been demonstrated with the use of packed columns; however, a recent observation [l] highlighted a problem regarding the use of a particular support material for this application.

In addressing this problem we sought to eliminate the support entirely by using an open tubular column and explored the use of one of the commercially available wide bore glass capillary columns with 0.75 mm internal dia- meter, as distinct from the fused-silica ‘Megabore’columns with an internal diameter of 0.53 mm. We have previously shown [2] that, provided there is sufficient attention to detail relating to the modified injection port assembly and introduction of sample, reproducible quantitative results may be obtained from analysis of the N-heptafluorobutyryl isobutyl ester (HBB) derivatives of amino acid standards.

The current study presents quantitative results from analyses of amino acid levels in plasma together with a comparison between these and similar data from corresponding packed column and FSOTcolumn analyses.

2 Experimental 2.1 Materials and Methods

Amino acid standard solution: The composition of the amino acid standard solution, apart from the inclusion of norleucine (10 mg/25 ml), was as previously described [3]. The amino acid concentrations are similarto thosefound in plasma and were selected in order to minimize errors during the quantitation of recovery experiments by adding amino acids to plasma.

lnternal standard solution: Norleucine (20 mg) was dissolv- ed in 0.25 M hydrochloric acid (50 ml). This solution was sub- divided into 10 ml aliquots and stored at 4OC for up to 12 months before renewal.

Esterifying reagent ( 3 ~ hydrogen chloride in isobutanol): This reagent was prepared as previously described [3].

Acylating reagent: Heptafluorobutyric anhydride (HFBA) was obtained from Fluka, Buchs, Switzerland, subdivided into small aliquots (0.6 ml) and stored under nitrogen in sealed vials at 4°C.

2.2 Sample Preparation

Pre-chromatographic treatment of plasma samples and subsequent derivatization of these and amino acid standards were carried out using previously described procedures [3]. Derivatives were stored in acetic anhydride at -2OOC. Samples for injection onto both packed and wide bore columns were drawn from this solution. For capillary analysis, a less concentrated solution was prepared by

0 1988 Dr. Alfred Huethig Publishers Journal of High Resolution Chromatography & Chromatography Communications 229

GC of Plasma Amino Acids

diluting aliquots (10 pl) with additional acetic anhydride (40 pl) and stored at -2OOC prior to use.

2.3 Chromatography

GC was carried out using two chromatographs, a Varian Model 3700 and a Carlo Erba Model 5360 Mega series. The latter incorporated a cold on-column injector and both were equipped with flame ionization detectors.

Analysis conditions were:

The Varian was fitted with i) a silanized coiled glass column ( 3 m X 2.7 mm i.d.) packed with 3.5%0V-l01 on resilanized Supelcoport (a pre-1984 batch of 100-1 20 mesh) and ii) a cross-linked apolar SPB-1 wide bore glass capillary column (0.75 mm i.d. X 30 m; 1 pm film thickness) linked to modified injector and detector ports with lengths of deactivated fused-silica. These modifications were accomplished with the aid of commercially available conversion kits.

Packed column: Injector temperature, 210OC; detector temperature, 250OC; carrier gas (nitrogen) flow, 19 ml/ min; hydrogen flow, 30 ml/min; air flow, 300 mlfmin. 1 pl samples were injected.

Wide bore capillary column: Injector temperature, 18OOC; detector temperature, 250OC; carrier gas (nitrogen) flow, 4 ml/min; make-up gas (nitrogen) flow, 30 mllmin; hydrogen flow, 30 ml/min; air flow, 300 ml/ min. 0.2 pl samples were injected.

For both columns, the oven temperature was pro- grammed from 100°C, after a post injection hold of 5 min, to 24OOC at 5°1min. After 1 min at 24OoC, the temperature was increased ballistically to 295OC at which it was held for 16 min prior to returning to 1 OOOC.

The Carlo Erba was fitted with an apolar DB-1 fused- silica column, 0.32 mm i.d. X 30 m; 1 pm film thickness (obtained from J & WScientific, Inc., Folsom, California, USA).

Detector temperature, 295OC; carrier gas (hydrogen) flow, 1.4 ml/min; air flow, 300 ml/min; hydrogen flow, 30 mllmin. After injection, the secondary cooling was immediately switched off and the initial oven temper- ature of 11 5OC was maintained for 8 min before being increased at 4Olmin to 170OC. The temperature was held constant for 3 min and then increased at 4O/min to 26OOC and ballistically at 40°/min to 285OC and held for 27 min before cooling to 11 5°C. 0.2 pl samples of the diluted derivatives were injected.

2.4 Statistics

Statistical evaluation of results was carried out using the two-tailed student's 't' test.

3 Discussion and Results In previous reports [3,4] a complete methodology, incorpo- rating a novel clean-up technique, for quantitative determination of amino acids in plasma by GC using packed and FSOT columns has been described. Because of the problems encountered in obtaining satisfactory support material [l], the use of a wide bore glass capillary column was evaluated usingamino acid standards [2] and it has now been extended to the quantitative analysis of amino acids in plasma. The use of wide bore columns result- ed in resolution superior to that of the packed column (Figures 1A and B), particularly in the region of methionine and phenylallanine which is clearly resolved from the flanking peaks.

Reproducibilities of below 12% and analytical recoveries of 77-1 22% (n = 6 of the same plasma sample) using the two columns are similar (Table 1) except for levels of aspartic acid and phenylalanine, the former being considerably higher using .the wide bore column, the reverse being true for the latter. These elevated values arise from spurious co-eluting peaks. Data from wide bore, packed, and FSOT column analyses are compiled in Table 2. Comparison of results from the wide bore with those from the packed column shows a significant difference (p < 0.005) between values for glycine, serine, isoleucine, proline, methionine, aspartic acid, phenylalanine, and lysine. In the case of glycine, seririe proline, and methionine, this arises from the inability of the packed column to resolve the amino acid peaks from iclosely eluting spurious peaks. As expected, these differences are not reflected in a similar comparison of wide bore data with those from the FSOT column. The difference in isoleucine figures is due to a small peak which co-elutes on the wide bore column but which is separated by reducing ,the flow rate (Table 2) leading to a result similar to that from the FSOT column.

By reducing the carrier flow rate to 2 ml/min and modifying the oven temperature program to include a three minute isothermal period at 190°C, chromatograms of the type shown in Figure 2 were obtained. Comparison with that in Figure 1 shows that the peak previously attributed to aspartic acid has been partially resolved into two, the later being identified as aspartic acid. The adjustment of analysis conditions, however, also leads to diminished resolution of phenylalaniine. Consequently, a quantitative assessment of both aspartic acid and phenylalanine using these conditions is only possible at levels as they occur in plasma (Table 2); the addition of these two amino acids to plasma for recovery purposes cannot be quantitated because of inadequate resolution. In addition, these adjustments in chromatographic conditions result (Table 2) in i) isoleucine and lysine levels which are in closer agreement with the corresponding figures from the FSOT column and ii) an apparently significant difference (p < 0.005) between

230 VOL. 1 1, MARCH 1988 Journal of High Resolution Chromatography & Chromatography Communications

GC of Plasma Amino Acids I

A

h

C

P

B

w cn z 0 P cn w U

P

I I I I 7- I I

0 10 2 0 3 0 0 10 20 3 0 Figure 1

A) Chromatogram of HBB derivatives of plasma amino acids obtained using a cross-lined apolar SPB-I wide bore glass capillary column (0.75 mm i.d. x 30 m; 1 pm film thickness).

B)Chromatogramof HBBderivativesof plasma amino acidsobtainedusinga silanizedcoiledglasscolumn (3m X 2.7 mmi.d.) packed with3.5%OV-l01 on Supelcoport (100-1 20 mesh).

Peak identification code: a, alanine; b, glycine; c, valine; d, threonine; e, serine; f, leucine; g, isoleucine; h, norleucine (internal standard) i, proline; j, pipecolicacid (internal standard); k, hydroxyproline; I, methionine; m, asparticacid; n, phenylalanine;~, glutamicacid; p, lysine; q, tyrosine; r, arginine; s, histidine.

I

T I M E I M I N S ) T I M E I M I N S )

Table 1

Reproducibility and recoveries of plasma amino acids (pmol/l) using wide bore and packed (bracketed) column analyses. ~

Amino acid

Alanine Glycine Valine Threonine Serine Leucine lsoleucine Proline Methionine Aspartic acid Phenylalanine Glutamic acid Lysine Tyrosine Arginine Histidine

Plasma Mean levelb) CV (%)') Level added

Plasma & added amino acidsa) Mean levelb1 CV (Oh)') Recovery (%)

~

300 (289) 232 (195) 206 (1 99) 193 (187) 99 (1 09)

114 (1 13) 75 (55)

110 (97) 25 (20)

103 (44) 60 (1 60)

491 (452) 181 (144) 57 (67) 66 (70) 95 (101)

3.2 (3.7) 6.7 (2.4) 3.9 (2.5) 4.6 (1.7) 2.7 (3.6) 2.0 (2.4) 5.7 (3.0) 4.5 (7.6) 9.2 (4.2)

24.5 (1 0.3) 7.2 (3.8) 6.1 (5.1) 2.3 (4.0)

10.9 (9.9) 6.2 (6.3) 3.9 (1 2.2)

449 266 171 168 38

152 152 174 27 30

121 272 274 110 115 129

752 (731) 485 (460) 375 (365) 351 (341) 133 (148) 262 (260) 220 (1 97) 281 (277) 56 (44)

193 (72) 207 (273) 723 (673) 436 (369) 1 59 (1 70) 159 (159) 234 (218)

3.0 (4.9) 3.6 (5.5) 3.0 (5.7) 3.3 (4.6) 5.9 (3.0) 2.1 (4.6) 2.6 (5.6) 1.9 (7.1) 7.9 (7.4)

30.3 (7.0) 4.8 (4.2) 4.2 (6.9) 6.5 (4.8) 2.8 (8.6) 3.3 (4.7) 4.6 (7.0)

100.7 (98.4) 95.1 (99.6) 98.8 (97.1) 94.1 (91.7) 89.5 (1 02.6) 97.4 (96.7) 95.4 (93.4) 98.3 (1 03.5)

114.8 (88.9) 300.0 (93.3) 121.5 (93.4) 85.3 (81.3) 93.1 (82.1) 92.7 (93.3) 80.9 (77.4)

107.3 (90.7)

a) 500 WI aliquots of plasma with 25 pI of amino acid standard solution added.

b, Levels represent the mean of six separate analyses of the same sample.

') CV = Coefficient of variation

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GC of Plasma Amino Acids

Table 2

Amino acid levels (pmol/l) in samples of the same plasma using wide bore column (under differing conditions) along with packed and FSOT columns.

~ ~~

Amino acid Wide bore Wide bore Packed FSOT (modified)a)

Mean SD Mean SD Mean SD Mean SD

Alanine 300 10 309 6 2!89 11 305 6 Glycine 232b) 15 236" 6 '195b,d) 5 223 8 Valine 206 8 209 5 199 5 210 4 Threonine 193 9 196d) 4 '187d' 3 198 7 Serine 99b) 3 9@) 3 .l09b,d' 4 104 3 Leucine 114 2 116 2 'I 1 3 3 113 1 lsoleucine 75bS') 4 53 1 55b) 2 51') 3 Proline 110b' 5 118dxe) 2 97b.d' 7 103e) 8 Methionine 25b' 2 32d) 3 20b.d' 1 27 2 Aspartic acid 103b2C) 25 44 1 44b' 5 43') 3 Phenylalanine 60b,') 4 67d) 4 160bzd) 6 75') 4 Glutamic acid 49 1 30 493 13 452 23 504 35

Tyrosine 57 7 57e) 4 67 7 47e' 3 Arginine 66 4 62@ 2 70d) 4 62 3

Lysine 18lb3'' 4 181d) 12 144b1d) 6 164') 10

Histidine 95C' 4 92 12 101 12 76') 4

a) Reduced carrier flow rate and isothermal period at 190°C

b, wide bore vs packed p < 0.005.

') wide bore vs FSOT p < 0.005.

d, wide bore (modified conditions) vs packed p < 0.005.

e, wide bore (modified conditions) vs FSOT p < 0.005.

tyrosine wide bore and FSOT data due to improved reproducibili,ty of the former value. In contrast, only partial separation olf histidine from a closely eluting substance on the wide bore column leads to a greater variation, and therefore resolution and quantitative analysis of histidine can only be satisfactorily accomplished using the FSOT column.

The recovery and reproducibility data obtained from plasma amino acids using the packed column (Table 1) is similar to that obtained in a previously reported analogous exercise [3]. In addition, the wide bore column obviates the problems associated with certain support materials and also provides satisfactory quantitative data after at least 600 injections, a performance comparable with the best packed columns produced in this laboratory.

1 Figure 2

Chromatograrn of HBB derivatives of plasma amino acids obtained usins - r , , 1 thewide borecolumnasinFig. 1, botwith reducedflowrateofcarriergas 0 10 20 3 0 and a 3 min. isothermal period at 190OC.

T I M E M I N S ) Peak identification as in Fig. 1.

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4 Conclusion

Reproducible analysis of plasma amino acid levels, using a wide bore glass capillary column, has been demonstrated. Compared with results from a packed column, the enhanced resolving power enables results to be obtained which are in closer agreement with those from the FSOT column. The wide bore column, therefore, is accepted as a suitable alternative to the packed column in this applica- tion, offering not only improved quantitative results, but also avoidance of troublesome support materials.

Acknowledgment

We thanksupelco Inc., Bellefonte, PAfor donating the SPB-1 wide bore glass capillary column and fused-silica links together with

both injection and detector conversion kits. We also thankSABAX SA for donating the Varian 3700 gas chromatograph.

References

Journal of High Resolution Chromatography & Chromatography Communications

1. M. Moodie, G. S. Shephard, and D. Labadarios, J. Chro- matogr. 362 (1986) 407.

1. M. Moodie, J. A. Burger, and D. Labadarios, J. High Resoln. Chrom. & Chrorn. Cornm. 10 (1 987) 383.

D. Labadarios, G. S. Shephard, E. Botha, L. Jackson, 1. M. Moodie, and J. A. Burger, J. Chrornatogr. (Biomed. Appl.) 383 (1986) 281.

D. Labadarios, 1. M. Moodie, .I. A. Burger, and G. S. Shephard, J. High Resoln. Chrom. & Chrorn. Cornm. 9 (1986) 555.

Ms received: November 9,1987 Accepted by REK: November 24,1987

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