Quantitation of xylose from plasma and urine by capillary column gas chromatography

CIinica Chimicu Acta, 137 (1984) 13-20 Elsevier

13

CCA 02743

Quantitation of xylose from plasma and urine by capillary column gas chromatography Stephen

L. Johnson

and Michael

Mayersohn

*

Department of Pharmaceutical Sciences, College of Pharmacy, The Unruersiiy of Arizona, Tucson. AZ 85721 (USA) (Received

August 22nd 1983)

Key words: Xylose; Monosaccharide; Pentose: Aldose; Methyloxime derivate

Summary A specific and sensitive assay for quantitation of xylose from plasma and urine has been developed. Following a clean-up procedure, plasma (0.1 ml) or urine (0.2 ml) samples are concentrated and undergo two sequential derivatization steps. A methyloxime derivative is formed initially, followed by trimethylsilylation of all hydroxyl groups, The derivatized samples are quantitated by capillary column gas chromatography using flame ionization detection. Xylose and the internal standard (2-deoxy-D-ribose) have retention times of 6.5 and 5.2 min, respectively. Other monosaccharides (e.g. ribose, arabinose) do not interfere with the assay. Standard curves are linear and reproducible over a concentration range of lo-200 mg/l for plasma and 100-2000 mg/l for urine. The within-day and day-to-day percentage coefficients of variation were less than 5 and 98, respectively, for plasma and urine.

Introduction D-Xylose is a monosaccharide that is employed clinically to assess gastrointestinal absorptive function. o-Xylose has traditionally been quantitated in biological fluids by calorimetric methods, with the Roe and Rice [l] procedure serving as the standard assay. The disadvantage of these assays lies in their non-specificity, since the same intermediates are formed by different monosaccharides. In the last decade more emphasis has been given to the quantitation of mono- and disaccharides by gas liquid chromatography (GLC). Few of these assays have been applied to biological fluids. Deneef [2], Wells et al [3], Bhatti et al [4] and Butts et al [5] have all described GLC techniques for analysis of sugars in blood and/or urine. * Address

correspondence

0009~8981/84/$03.00

to this author.

0 1984 Elsevier Science Publishers

B.V.

14

In each case. trimethylsilyl (TMS) ethers of mono- and disaccharides were formed. Such an approach yields several peaks for each carbohydrate due to the derivatization of multiple anomers. These procedures result in a lower signal to noise ratio. decreased sensitivity. and poorer resolution than if each saccharide produced a single peak. A complex chromatogram results and quantitation based on measuring the area produced by one anomer must assume that the ratio of anomers remains constant. Further disadvantages of these methods include the necessity of using large volumes of reagents [2,3]. extensive clean-up procedures (i.e. prior fractionation via ion exchange) [5], and absence of validation of the methods for application to pentoses [2-51. Horning et al [6], Heaf et al [7] and Murphy et al [8] have also described GLC methods for the analysis of carbohydrates in urine or plasma. In each of those methods an oxime or methyloxime derivative was formed to decrease the multiplicity of peaks caused by anomerization. A second derivative was then formed prior to actual chromatography of samples. These methods with the exception of Murphy et al are not well suited for analysis of large numbers of samples, as they require large sample volumes as well as lyophilizing large volumes of aqueous reagents [6]. and generally extensive preparatory clean-up procedures prior to derivatization [6.7]. The Murphy and Pennock [8] method for analysis in blood seems rapid and utilizes small sample volumes: however, the focus was on glucose determination. Although retention times for pentoses were given, no validation data for these compounds were presented. Storset et al [9] have developed an assay for monosaccharides in seminal fluid, in which methyloxime-TMS derivatives are separated on a glass capillary column coated with SE-30. The methyloxime-TMS derivatives are more volatile than the acetate derivatives prepared by Murphy et al [8] and thus are expected to have shorter retention times [lo]. We have applied capillary column GLC to the quantitation of D-xylose in both plasma and urine. The compounds are separated as the more volatile methyloximeTMS derivatives. We report here the validation of our method and illustrate its application to an examination of xylose disposition in man. Materials and methods Reagents Acetonitrile and methanol were obtained from Burdick and Jackson (Muskegan, MI, USA). Methoxylamine-HCl, silylation grade pyridine, and BSTFA were purchased from Pierce Chemical Co. (Rockford, IL, USA), Mallinckrodt Amberlite XAD-2 resin was purchased from American Scientific Products (Phoenix, AZ, USA). Standards D-Xylose, D-ribose, D-arabinose and 2-deoxy-D-ribose were obtained from Sigma Chemical Co. (St. Louis, MO, USA). D-Xylose for oral dosing (Xylophan) was obtained from American Scientific Products (Phoenix, AZ, USA).

15

Plasma standards and controls. Blank plasma was obtained from the Arizona Health Science Center blood bank. o-Xylose in distilled water was added to plasma to provide standard concentrations. Concentrations of 10, 20, 50, 100, 150 and 200 mg/l were prepared for the standard curve, while concentrations of 15, 125, 190, and 1900 mg/l were employed as controls for validation purposes. Urine standards and controls. Samples for standard curves were prepared by the addition of o-xylose in distilled water to human urine, producing concentrations of 100, 200, 500, 1000, 1500, and 2000 mg/l. Controls for assay validation were prepared at concentrations of 150, 1200, 1900, and 15,000 mg/l. Procedures Plasma samples Plasma samples of 100 ~1 in 10 X 75 mm tubes were mixed with 500 ~1 of acetonitrile by vortex mixing for 10 s. A loo-p1 volume of aqueous internal standard solution, 2-deoxy-o-ribose (100 mg/l) was pipetted into each tube, and samples were vortexed for an additional 15 s. Samples were then centrifuged at 3000 rpm for 5 min and the protein-free supernatant was transferred to clean 10 mm X 75 mm disposable culture tubes. Samples were evaporated to dryness under nitrogen at 65°C. The residue was reconstituted in 700 ~1 methanol and tubes were vortexed for 15 s to assure good mixing. The solution was transferred to a 1 ml Reactivial (Pierce Chemical Co., Rockford, IL, USA) and evaporated to dryness under a gentle nitrogen stream at 60°C. Samples at this point may be stored in a refrigerator overnight without affecting the final results. A 40-~1 volume of methoxylamine-HCl in pyridine (25 mg/ml) was added to each Reactivial. The vials were capped and vortexed for 30 s prior to being placed into a heating block at 80°C for 30 min. Samples were allowed to cool for 10 min and then 60 ~1 of BSTFA was added to each vial. The vials were capped, vortexed for 10 s and placed in a heating block at 70°C for an additional 30 min. Samples were cooled and 0.5~~1 aliquots were injected into the gas chromatograph. Urine samples A 200~~1 volume of urine preserved with 1% sodium fluoride (w/v) was transferred to a clean 10 X 75 mm culture tube along with 200 ~1 of 2-deoxy-D-ribose as the internal standard (1000 mg/l). Tubes were vortexed for 10 s and then the sample was passed over Amberlite XAD-2 resin packed into a l-ml plastic syringe. Water was passed through these columns prior to the passage. of sample in order to wet the resin. The columns were suspended in culture tubes (13 X 75 mm) and centrifuged at 3000 rpm for 5 min. A lOO-~1 volume of the eluate was collected and transferred to clean 10 x 75 mm culture tubes. The sample was evaporated to dryness under nitrogen at 65°C. The residue was reconstituted in 700 ~1 methanol and the tubes were vortexed for 15 s. The residue layer was broken up with a Pasteur pipette to insure complete dissolution of xylose and the internal standard. Tubes were centrifuged for 5 min and the supernatant was transferred to clean 1 ml Reactivials. The

16

solution was evaporated to dryness under a gentle stream of nitrogen at 60°C. The samples were then derivatized in the same manner as described above for the plasma samples. Gas chromatograph_v

Derivatized samples were analyzed using a Hewlett-Packard 5840A gas chromatograph equipped with a 5840A integrator, a capillary inlet system, and an FID detector (Hewlett Packard, Avondale, PA, USA). The column employed was a Hewlett Packard 25-m fused methylsilica column. An injection port temperature of 350°C and a detector temperature of 300°C were maintained. The carrier gas was helium and the column pressure was maintained at 23 psi. The makeup gas flow rate was 45 ml/min and the hydrogen and air flows were 30 ml/min and 240 ml/min, respectively. A trilevel temperature program was employed for plasma samples, and all injections were made in the splitless mode. The initial oven temperature was 120°C which was immediately increased to 160°C at a rate of 30 degrees/min. After 4 min total elapsed time from injection, the temperature was increased to 20°C at a rate of 11 degrees/min. After 7.8 min total elapsed time the temperature was again increased to 260°C at a rate of 30 degrees/min. A bilevel temperature program was used for the urine analysis with a column split ratio of 10 to 1. All gas flows were identical to those given above. The injection port temperature was lowered to 250°C in order to minimize the appearance of pyrolysis products on the chromatogram. The initial oven temperature was held at 160°C where it was maintained for 4 min. The oven temperature was then raised to 220°C at a rate of 16 degrees/min. At 7.5 min total elapsed time the temperature was further increased to 260°C at a rate of 30 degrees/min. Xylose concentrations in plasma and urine were determined from a standard curve of the ratio of areas (xylose/internal standard) vs. xylose concentration. Results and discussion The derivatization and gas chromatographic procedures described here resulted in separation with baseline resolution of the three closely related pentoses D-ribose, D-arabinose and D-xylose. A typical chromatogram (Fig. 1) illustrates the separation achieved by this procedure from an aqueous sample. Fig. 2A depicts a chromatogram from blank plasma, to which no sugars have been added. There are no visible peaks in the area of the internal standard (5.2 min) or at the retention time where xylose normally appears (6.5 min). In over 25 blanks collected from different subjects, the region of the chromatogram was devoid of peaks where the internal standard normally appears, thus supporting our choice of internal standard. Fig. 2B shows a chromatogram obtained from analysis of a plasma sample collected 4 h after one subject had ingested a 25-g oral dose of xylose. The concentration in this sample was determined to be 180 mg/l. The procedure employed results in a single peak for the internal standard and two peaks for xylose, a minor peak followed immediately by a much larger peak. While on a packed column these peaks are usually not

A

-

4

Time,

.A

__

68

_-2.._I_a_

2

2 min

4 6 8 Time, min

Fig. 1. Gas chromatogram illustrating the separation of xylose from several closely related pentose An aqueous sample was spiked with xylose (X), arabinose (A) and ribose (R).

10

sugars.

Fig. 2. (A) Gas chromatogram of a blank plasma sample. (B) Gas chromatogram of an authentic plasma sample containing xylose (X, 180 mg/l) and the internal standard, 2-deoxy-D-ribose (D. 100 mg/l). The plasma sample was obtained from one subject 4 h after a 25-g oral dose of xylose.

resolved, this is not always the case on a capillary column. The two xylose peaks apparently represent the syn- and anti- forms of the me~yloxime, where one form greatly predominates over the other [llf. We have found that by measuring the larger peak the presence of the minor peak has no effect on the assay reproducibility. Fig. 3 shows a chromatogram from blank urine and a chromatogram from the analysis of a urine sample collected between 8 and 10.5 h after a 25-g oral dose of xylose was ingested by one subject. The retention times are similar to those reported here for plasma. The baseline for blank urine samples can be somewhat variable. In B

A

&_d.LL L-L-.

0

I

2

6 8 4 Time, min

10

_ u_ 0

i *ii ii_i_(._-_ 2 4 6 8 10 Time, min

Fig. 3. (A) Gas chromatogram of a blank urine sample. (B) Gas chromato~am of an authentic urine sample containing xylose (X, 1690 mg/l) and the internal standard, bdeoxy-D-ribose (D, 1000 mg/l). The urine sample was obtained from one subject during a colfeetion period S-10.5 h after a 25-g oral dose of xyiose.

IX

general, an overnight sample has much more baseline noise than samples collected later in the day. Table I shows the reproducibility and recovery data for both plasma and urine at 4 different concentrations. Since xylose is generally found in much higher concentrations in urine than in plasma, the assay was validated in urine at higher concentrations. The within-day and day-to-day percentage coefficients of variation were not greater than 5 and 9%, respectively for both types of biological fluids. As previously noted, the baseline associated with a chromatogram of urine can be somewhat variable. It is difficult to clean-up a urine sample thoroughly. Xylose being highly polar cannot be extracted from the aqueous samples. Since the components of urine consist, for the most part, of highly polar compounds, these compounds are also not easily extracted. We found that passage of urine through Amberlite XAD-2 resin resulted in a visibly cleaner sample. Furthermore, when this step was omitted the derivatized sample forms a solid at room temperature. It was noted above that a trilevel temperature program was employed for plasma samples, while a bilevel program was used for urine samples. The type of programming used is a function of the injection mode. In the splitless injection mode, the amount of solvent reaching the column requires the use of lower initial oven temperatures. It is recommended that initial temperatures be below the boiling point

TABLE

I

Within-day

and day-to-day

No. of samples

variation

of the xylose assay

Xylose concentration, added

cv

(%)

Recovery

mg/l determined

(SD) (0.5) (4.5) (9.0) (60.0)

3.7 3.8 4.9 3.1

94.1 94.6 96.4 101.9

Within-day plasma

10 10 10 10

15 125 190 1900

14.2 118.3 183.1 1935.9

urine

10 10 10 10

150 1200 1900 15.000

148.8 1187.4 1866.0 13.877.1

(5.4) (35.6) (76.5) (555.1)

2.9 3.0 4.1 4.0

99.2 99.0 98.2 92.5

plasma

6 6 6 6

15 125 190 1900

15.2 121.6 188.0 1949.3

(1.2) (3.5) (4.0) (122.8)

7.9 2.9 2.1 6.3

101.3 97.3 98.9 102.6

urine

5 5 5 5

150 1200 1900 15,000

148.6 1255.6 1894.0 15.355.4

(13.4) (65.3) (126.9) (644.9)

9.0 5.2 6.7 4.2

99.1 104.6 99.7 102.4

Day-to-day

(% )

19

of the injected solvent. If the splitless mode is used at higher temperatures, we found peak shapes to be distorted. The splitless mode was employed for analysis of plasma samples to permit use of relatively small sample volumes. A trilevel temperature program was used in order to accommodate the restrictions imposed by the splitless injection mode. While the initial oven temperature was well above the boiling point of the solvent injected, it was low enough to insure good peak shape without the excessive turnaround time necessary when the oven is cooled dramatically. Huguenin et al [12] have examined the disposition of xylose in man using the Roe and Rice [l] calorimetric assay. There was an apparent dose-dependence seen in the terminal elimination half-life. When lower doses of xylose are given, it becomes necessary to measure lower plasma concentrations in order to obtain an estimate of half-life. Those authors suggested that the apparent dose-dependent half-life might be an artifact of the non-specific nature of the assay at lower concentrations. Fig. 4 illustrates the application of the present method. This figure is a xylose plasma concentration-time profile after one subject received a 5-g intravenous dose of xylose as a 15-min infusion. It is of interest to note that even if one allows for twice the reported assay sensitivity (approximately 100 mg/l) of most of the calorimetric assays (13-16) a considerable portion of the terminal phase would not be measurable, and a much different terminal half-life would have been measured. We have presented here a GLC assay for D-xylose in both plasma and urine. Both the reproducibility and recoveries are acceptable for analytical work. While other GLC assays have been proposed few have been applied to biological fluids. Perhaps one of the best assays to date for biological fluids was that of Murphy and Pennock [8]; however, the emphasis of that assay was given to glucose and no validation was presented for pentoses. While the present assay may be too time consuming for use clinically, it may have application as a specific reference method, to which the currently used clinical assays may be compared. Such large scale comparisons involving individuals who have received doses of xylose are currently in progress.

10' 1

2

3

5

6

Time,4h Fig. 4. Xylose plasma g) to one subject.

concentration

as a function

of time after a 15-min intravenous

infusion

of xylose (5

Acknowledgement This study Aging.

was supported

by Grant

AGO3460

from

the National

Institute

on

References 1 Roe JH. Rice EW. A photometric method for the determination of free pentosea in animal tissues. J Biol Chem 1948; 173: 507-512. determination of mixtures of monosaccharides, disaccharides. and alditols as 2 Deneef J. Quantitative their trimethylsilyl ether derivatives by gas-liquid chromatography. Chn Chim Acta 1969; 26: 485-490. analysis of serum and urine sugars by gas chromatography. 3 Wells WW, Chin T, Weber B. Quantitative Clin Chim Acta 1964; 10: 352-359. 4 Bhatti T. Clamp JR. Identification and estimation of monosaccharides and disaccharides in urine by gas-liquid chromatography. Clin Chim Acta 1968; 22: 5633567. identification of urinary carbohydrates Isolated by anion5 Butts WC. Jolley RL. Gas-chromatographic exchange chromatography. Clin Chem 1970: 16: 722-725. 6 Horning EC. Horning MG. Plasma sugars and sugar alcohols. Methods Med Res 1970; 12: 400%401. 7 Heaf DJ, Gaiton DJ. Sorbitol and other polyols in lens. adipose tissue and urine in diabetis mellitus. Clin Chim Acta 1975: 63: 41-47. measurement of blood and urine glucose and other 8 Murphy D, Pennock CA. Gas chromatographic monosaccharides. Clin Chim Acta 1972; 42: 67-75. and monosaccharide derivatives in human seminal 9 Storset P, Stokke 0. Jellum E. Monosaccharides plasma. J Chromatog 1978; 145: 351-357. of trimethylsilyl deriva10 Sweeley CC. Bentley R. Makita M, Wells WW. Gas liquid chromatography tives of sugars and related substances. J Am Chem Sot 1963; 85: 249772507. analysis of sugars and related hydroxy acids as acyclic oxime and 11 Petersson G. Gas-chromatographic ester trimethylsilyl derivatives. Carbohyd Res 1974: 33: 47-61. du D-xylose. Schweiz Med Wschr 12 Huguenin PH. Cochel B. Balant L. Loizeau ED. Test d’absorption 1978; 108: 206-214. L. A new simple ultramicromethod for the determination of D-xylose in 13 Rozental M, Tomaszewski blood and urine. Clin Chim Acta 1974; 50: 311-317. micromethod for xyloae in 14 Eberts TJ, Sample RHB. Glick NLR. Ellis GH. A simplified calorimetric serum or urine with phloroglucinol. Clin Chem 1979; 25: 1440-1443. method for blood and urine xylose. Clin Chim 15 Buttery JE, Kua SL. Dewitt GF. The ortho-toluidine Acta 1975: 64: 325-328. of xylose in plasma. Analyst 1975; 100: 12-15. 16 Trinder P. Micro-determination