Life Sciences, Vol. 29, pp. 1689-1696 Pergamon Pres Printed in the U.S.A.

DETERMINATION OF 3-METHOXY-4-HYDROXYPHENYLGLYCOL IN URINE BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

WITH AMPEROMETRIC DETECTION

Rafael Alonso*, Candace J. Gibson** and James McGill

Laboratory of Neuroendocrine Regulation Department of Nutrition and Food Science Massachusetts Institute of Technology

Cambridge, Massachusetts 02139 *Departamento de Fisiologia y Bioquimica

Facultad de Medicina Universidad de La Laguna

La Cuesta, Tenerife (Canary Islands)

(Received in final form August 20, 1981)

Summary

A reversed-phase high-performance liquid chro- matographic method has been used for the quantitative determination of 3-methoxy-4-hydroxyphenylglycol (MHPG) in urine. After incubation with glusulase, free MHPG is extracted into ethyl acetate and further isolated by a combination of thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The addition of amperometric detection provides increased sensitivity to a highly specific assay.

The major norepinephrine metabolite in the mammalian nervous system is 3-methoxy-4-hydroxyphenylglycol (MHPG) and its sulfated conjugate (MHPG-SO 4) (1,2). MHPG appears in animal and human urine as glucuronide and sulphate conjugates (3-5). The con- tribution of peripheral versus brain sources of these urinary metabolites remains controversial (6-11); however, the measure- ment of MHPG in human urine has been used as an index of CNS norepinephrine metabolism in some psychiatric diseases, most notably the affective disorders (12-14), or as a predictor of response to antidepressant drug therapy (15).

Since current fluorometric methods are not specific enough for the assay of MHPG in urinary samples, its determination has usually been achieved by gas chromatography (5,16,17) or gas chromatography with mass spectrometry (18). This report presents a method for the determination of urinary MHPG using high- performance liquid chromatography (HPLC) with amperometric detection.

**Author to whom reprint requests should be sent.

0024-3205/81/161689-08502.00/0 Copyright (c) 1981 Pergamon Press Ltd.

1690 Urinary MHPG Measurement by HPLC Vol. 29, No. 16, 1981

Materials and Methods

Apparatus and liquid chromatographic conditions. The liquid chromatographic system (Bioanalytical Systems, W. Lafayette, IN) consisted of a column, precolumn, amperometric detection and pulse dampening unit (LC-4, Bioanalytical Systems), single-piston pump (Model IIOA, Altex Scientific Co., Berkeley, CA), sample injector and chromatogram recorder. The stainless steel ~column (30 cm x 3.9 mm) was packed with microparticulate reversed-phase material (~Bondapak C18 , particle size i0 ~m; Waters Assoc., Milford, MA). A short precolumn (2 x 69 mm) with reversed-phase particles (30 um, Whatman Inc., Clifton, NJ) protected tile column. The detector (model TL-3) was packed with CP-O carbon paste (Bioanalytical Systems). Sample injection was accomplished with an automatic sample injector (WISP 710A; Waters Assoc.). Chromatograms were recorded and analyzed with a model 3385A Automation System (Hewlett-Packard Instruments, Lexington, MA).

The mobile phase, 0.05 M sodium phosphate, monobasic, buffer (pH 5) was made in distilled, deionized water (Millipore Q System, Millipore Corp., Bedford, MA) and filtered (0.45 ~m Millipore filters; Millipore Corp.) and degassed before use.

The system was run at a flow rate of 1.5 ml/min. The detector potential was set at +0.70 V versus a Ag/AgCI reference electrode.

Reagents. The following chemicals and reagents were used: MHPG-piperazine salt (Sigma Chemical Co., St. Louis, MO); MHPG-SO 4 (K + salt, Fluka AG Buchs SG; Switzerland); ~-glucuronidase/aryl sulfatase (glusulase) from Helix pomatia (B grade; Calbiochem- Behring Corp., La Jolla, CA); ethyl acetate (distilled in glass; Burdick and Jackson Laboratories Inc., Muskegan, MI); creatinine ZnCI (Sigma Chemical Co.). HPLC-grade methanol and isopropanol were used (Fisher Scientific Co., Pittsburgh, PA) as well as Analytical reagent-grade sodium acetate, sodium phosphate, mono- basic and hydrochloric acid (Baker Analyzed Reagents, Phillips- burg, NJ). Other chemicals and solvents used in the thin-layer chromatography (TLC) were reagent grade from commercially available sources.

Procedure. Twenty-four hour urine samples from laboratory personnel or student volunteers (i0 men; 2 women, ages 24-33 years) were collected in plastic bottles containing 5% sodium metabisulfite in 6 N HCI (approximately 1% of the total urine volume). Aliquots of 10-20 ml were adjusted to pH 3 with 6 N HCI and stored at -20°C until time of assay.

After thawing at room temperature, 0.25 ml of urine was placed in 12 x 75 mm disposable glass tubes with an equal volume of 0.5 M sodium acetate buffer (pH 6.5) containing 4 mg/ml of EDTA. Twenty ~l of glusulase were added and the samples were incubated at 37°C for 20-24 h (4). After cooling, i00 mg of sodium chloride was added, and free MHPG was extracted into 4 ml of ethyl acetate. After brief centrifugation to separate the phases, 3 ml of ethyl acetate was washed with 0.3 ml of 0.5 M KHCO 3. After further centrifugation, 2 ml of the ethyl acetate was dried with 50 mg of anhydrous sodium sulfate or, alternatively, passed through phase separating filters (Brinkman Instruments Inc., Westburg, NY).

Vol. 29, No. 16, 1981 Urinary MHPG Measurement by HPLC 1691

The ethyl acetate samples were evaporated to dryness using a rotating dryer (Speed Vac Concentrator, Savant Instruments Inc., Hicksville, NY), attached to a VirTis Lyophilizer (The VirTis Company, Inc., Gardiner, NY).

The dried residue was redissolved in 50-75 ~I of methanol and applied to TLC plates (20 x 20 cm, Silica gel-coated plate, LK5D; Whatman Inc.). The plates were developed to I0 cm (about 45 min) using the organic (bottom) layer of a chloroform:methanol:acetic acid:water (120:20:30:25) mixture. Pure standards of MHPG (i ~g) applied to the side channels of the plate were identified as a blue spot after spraying with Folin-Ciocalteau's phenol reagent (Sigma Chemical Co.). A segment corresponding to the location of authentic MHPG (Rf = 0.60) was scraped from the plate and eluted with 1 ml of filtered HPLC-grade methanol. Aliquots of 0.5 ml were evaporated to dryness and the residue was dissolved in 250 ~i of the mobile phase. Usually 50-109 ~I of the sample was injected into the LC system.

To determine the MHPG content of the original samples from the peak area on the chromatogram, a urine pool was made from the samples to be analyzed. Different amounts of pure MI{PG-SO 4 were added to 0.25 ml samples of the pool and were run through the whole procedure. The same pool, without the addition of MHPG-SO 4, was run every I0 samples. The concentration of MHPG in the pooled urine was calculated from the internal standard curve by plotting peak area (corrected for detector response) against the con- centration added to the pool and extrapolating to zero the area corresponding to the original pool. The concentration of MHPG in the unknown samples was calculated according to the equation:

MHPG (~g/ml) = Peak area of unknown x concentration x 4

Peak area of pool of pool

Peak heights were equivalent to peak area and could be used for calculations.

Calibration of the Hewlett-Packard integrator was made using an external standard, i.e., a known amount of free ~PG. To avoid variation in detector response, calibration was repeated every 25-30 samples.

Urinary creatinine was measured colorimetrically using a modification of Jaffe's method (19).

Results

The combination of solvent extraction with TLC isolation, and an LC system allows clear separation of MHPG from other inter- fering materials present in urine. In normal subjects, typical chromatograms show a retention time of 15.8 min for MHPG (Fig. i) corresponding to that of authentic MHPG (Fig. 2). Mean 24 h urinary MHPG excretion in twelve samples collected under non- controlled conditions was 2.65 + 0.20 mg/day (range = 1.94-4.11 mg/day). The two women excreted less MHPG, 2.20 mg/day as com- pared to 2.75 mg/day for the ten men. However, when expressed as mg MHPG excreted/g creatinine, the women's values were higher (since they excreted less creatinine per day), 2.03 + 0.37 mg MHPG/g c rea t in ine versus 1.50 ~ 0.15 mg/g creat inineT

1692 Urinary MHPG Measurement by HPLC Vol. 29, No. 16, 1981

F~ont

0 4 8 12 t6 20 Min

FIGURE i.

Typical Chromatogram of Human Urine Sample

Aliquots (0.25 ml) of a pooled urine sample were pro- cessed as described in text. Fifty ul of the final solution (250 ~i) were injected (A). Pure MHPG-SO 4 (0.5 ~g) was added to the second sample (B) and pro- cessed in the same way. Chromatographic conditions: C18 reversed-phase column; 0.05 M sodium phosphate buffer (pH 5); 1.5 ml/min flow rate; electrochemical detector +0.7 V; ambient temperature.

MHPG

InA

Front

I i I I 116 I 0 4 8 12 20

M i n

FIGURE 2.

Chromatogram of Authentic MHPG

The peak represents 5 ng of authentic MHPG (as pipera- zine salt) run directly over the C18 reversed-phase column under the same chromatographic conditions as in Fig. I.

Vol. 29, No, 16, 1981 Urinary MHPG Measurement by HPLC 1693

A typical internal standard curve of added MHPG-SO 4 is shown in Figure 3. The assay is linear up to i0 ~g added to 1 ml of urine and the sensitivity was i00 pg per injection. The inter- assay variation was 12.5% as determined on duplicates of nine samples run 3 weeks apart. The within day variation was 4.3~ as determined on twelve duplicate samples.

2 0 0

J:

<D

I00

50

....-" ,, I i I

0.5 1.0 2.0

M i c r o g r a m s M H P G - S O 4 A d d e d

FIGURE 3.

Internal Standard Curve

Various amounts of MHPG-SO 4 were added to 0.25 ml of a pooled urine sample and run through the whole procedure (see text). The abscissa represents the peak height (mm). All samples were run under the same chromatographic conditions as in Fig. i. Each point is the result of duplicate analyses.

Occasionally a small additional peak appeared slightly after, but well separated from, MHPG (retention time = 20 min). This peak may have been a contaminant from the glusulase preparation or from one of the organic solvents since it also appeared in urine blank samples (i.e., enzyme only run through the analysis procedure). Many types of HPLC-grade ethyl acetate were unsuit- able for use due to the presence of contaminating peaks (20). Both "Baker Analyzed" HPLC Reagent (J.T. Baker Chemical Co., Phillipsburg, NJ) and Malltnckrodt Analytical reagent grade ethyl acetate (Mallinckrodt Inc., Paris, KY) gave peaks which were close to or coincident with the MHPG peak. The Jackson and Burdick brand was the most acceptable and "cleanest" preparation.

Retention times of ~PG varied from column to column and with column age and therefore must be determined for each system (for instance, after 6 months use of one column, the retention time for MHPG had decreased from 15.8 min to 12 min). Using an LC-23 column heating apparatus (Bioanalytical Systems), the retention time of NHPG was decreased to 11.7 min at 40°C, enabling the

1694 Urinary MHPG Measurement by HPLC Vol. 29, No. 16, 1981

faster running of samples. Adding 5% methanol to the buffer resulted in a decrease in retention time to the same extent. When methanol was added to the mobile phase the carbon paste electrode was surfaced with CP-W paste (Bioanalytical Systems) to give a more stable baseline.

Discussion

High performance liquid chromatographic systems with electro- chemical, ultraviolet or fluorescence detection have, in many research laboratories, replaced the routine use of the much less sensitive fluorescence assays to measure catecholamines and their metabolites in tissue samples and biological fluids. These systems represent a simple [often samples can be injected directly on the LC system after precipitation of protein (21)], sensitive (pmole to fmole range), and relatively inexpensive innovation in the research laboratory.

There has been great interest in measuring urinary MHPG as a possible index of CNS norepinephrine function (12-14) and hence a sensitive, specific HPLC assay is necessary. The present method involving liquid extraction, TLC isolation, LC and amperometric detection provides a high degree of specificity and sensitivity to the measurement of MHPG in urine. Many substances contained in human urine are extracted in organic solvents at neutral pH and are detectable by the amperometric detector. Therefore, samples must be purified and MHPG isolated from other interfering substances. Thin-layer chromatography provides good separation and its combination with reversed-phase liquid chromatography gives an absolute resolution of urinary MHPG from unknown com- pounds (Fig. I).

Two recently published methods using LC systems with ampero- metric detection (22) and fluorescence detection (23) have slight drawbacks as compared to the present method. Although a simpler clean-up is involved in both procedures, the time gained in preclean-up steps is lost in the longer time necessary for the elution of the many peaks present in the simple ethyl acetate extracts (22). With the present method, using either a 5~ metha- nol mobile phase or a column heating unit, samples can be run once every 12-15 min. A fluorescence detector is far more expensive than an amperometric detector, but if already available seems a reasonable alternative. The sensitivity of the two methods is comparable (23).

Human urine contains free MHPG as well as sulphate and glucur- onide conjugates. Only the free compound is suitable for quanti- ration by the amperometric detector; therefore, determination of total urinary MHPG requires the hydrolysis of its conjugates. Even though we have limited the description of our method to that for total MHPG, tentatively each fraction can be measured from the same urinary sample; free MHPG would be extracted before enzymic hydrolysis and more specific enzyme preparations could be used (e.g., sulfatase alone for SO 4 conjugates)(4).

Enzymic hydrolysis of conjugates is very mild compared to acid hydrolysis, which may result in further degradation of the resulting free MHPG. Although commercially available glusulase preparations may contain small amounts of MHPG, this can be

Vol. 29, No. 16, 1981 Urinary MHPG Measurement by HPLC 1695

corrected by running enzyme blanks with each assay. Normally we find no measurable MHPG peak in enzyme samples run under the same conditions.

The sensitivity of the assay allows the use of small sample volumes (suitable for csf samples), saving time in the evapora- tion steps. Even though the recovery is slightly lower than in other methods (13,14,16), it is highly consistent and sufficient for the sensitivity of this method. As an additional advantage over other gas chromatographic methods, the present procedure does not require previous derivatization steps and special care with sample stability is not necessary. The assay has been used for human and rat urine with satisfactory results. As shown in Table I, the values obtained with the present method are in excellent agreement with those reported previously.

TABLE I

Typical Values of Total Urinary MHPG in Normal Human Subjects According to Different Methods a

Method Reference mg MHPG/24 h ~ g MHPG/mg creatinine

GC Bond and Howlett(5) 2.41 + 0.19

GC Bond(16) 1.84 + 0.13

GLC Cymerman and Francesconi(9)

GCMS Murray et al.(18) 2.62 + 0.34

HFLC-ED This method 2.65 + 0.20

(7) b 1.55 + 0.22

(9) 1.09 + 0.i0

(6) 0.74 + 0.04

(7) 1.53 + 0.12

(12) 1.50 + 0.15

aData are given as mean + SEM calculated from the individual values given in the original report.

bNumber of subjects.

Acknowledgements

These studies were supported in part by grants from the National Institutes of Health (AM-14228) and the National Aeronautics and Space Administration (NGR-22-009-627) to Dr. Richard J. Wurtman.

The authors thank C.J. Watkins and Y. Osaki for technical assistance and helpful comments and Dr. R.J. Wurtman for his continuous support and interest.

1696 Urinary MHPG Measurement by HPLC Vol. 29, No. 16, 1981

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