Sensitive assay for melatonin in human serum by liquid chromatography

ANALYTICA

CHIMICA ACIA

ELSEVIER

Analytica

Chimica

Acta 316 (1995) 377-385

Sensitive assay for melatonin in human serum by liquid chromatography Aldo Lagan’a a,*, Aldo Marino a, Giovanna Fago a, Beatriz Pardo-Martinez Mariano Bizzarri ’

b,

a Department of Chemistry, “La Sapienza” University of Rome, Pus. le Aldo More 5, Rome 00185, Italy b Hospital Central SW de PetvXeos, Mexicanos Perifkrico Sur 4091, M&co City, D.F. 14140, Mexico ’ Chair of Microsurgery, “Tor Vergata” Uniuersily of Rome, Rome, Italy

Received 9 February 1995; accepted25 May 1995

Abstract A rapid, sensitive and specific assay was developed for routine determination of melatonin in human serum. Melatonin was measured by liquid chromatography with fluorescence detection (A, = 285 nm, A,, = 345 nm), using an isocratic mobile phase consisting of water-acetonitrile (75:25, v/v). A simple “solid phase extraction” procedure employing LC-18 and Carbograph as adsorbents was used to isolate and purify the compound of interest from serum samples. The average recovery was 88.9% (range 86.3-91.7%). The method gave satisfactory reproducibility and accuracy, the detection limit of the system was 2 pg for injection and the practical limit of the assay for serum is ca. 3 pg ml-’ for 2 ml of sample. In order to evaluate the specificity, the potential interference of 33 compounds (chiefly monoamines) arising from the metabolism of tyrosine and tryptophan were studied. The melatonin concentration in 21 serum samples measured in the present study was compared by using an radioimmunoassay method. Some results on the melatonin circadian rhythm are presented to illustrate the applicability of this method. Keywords:

Melatonin;

Liquid chromatography;

Fluorimetry;

Circadian

rhythm

1. Introduction Melatonin is a circulating chief hormone synthesized by the enzyme N-acetyltransferase (NAT) which catalyzes the N-acetylation of serotonin to N-acetylserotonin. The latter is then further O-methylated to melatonin by the enzyme hydroxyindole0-methyltransferase (HIOMT) [1,2] (Fig. 1) in the pineal gland.

* Corresponding

author.

0003-2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0003-2670(95)00298-7

Investigation of the physiological functions of the pineal gland relies heavily on determination of serum melatonin concentration. This compound, which is also produced by extrapineal tissues, is believed to influence reproductive functions in a wide variety of species [3-51, the diurnal activity rhythms [6] as well as the immune system [7,8]. Aspects of the melatonin rhythm, which may be responsive to changes in the day/night cycle include the duration of the night-time increase, the total amount secreted and the timing of the peak concentrations [9].

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The temporal changes in melatonin availability as a consequence of alterations in its rhythmic production have also been strongly implicated in reproductive malfunctions in neurological and psychiatric disorders [lo-121. Over the past few years, melatonin has been postulated to be a potential sleep-inducing mediator. A number of investigations have pointed to an apparent sensitivity of pineal glands to other envi-

ronmental factors such as electric or weak, static, magnetic’ fields 113-161. There is now substantial evidence that alterations in the circadian rhythm or the amount of pineal melatonin secretion can increase the risk for cancer in laboratory animals [17,18]. Altered melatonin secretion has also been associated with certain forms of prostate and breast cancer in humans [19-221. Indeed, melatonin itself exhibits oncostatic properties

Tryptophan

SOH-Tryptophan

5OH-Tryptamine

N-acetyltryptamine

H3ce~q-q-~p

Melatonin

I

H Fig. 1. Tryptophan

metabolism

and melatonin

synthesis.

A. Lagad

et al./Analytica

versus certain cancer cell lines including carcinomas and breast cancer in vitro [23,24]. A number of reports containing somewhat contradictory results have been published on the levels of melatonin in cancer patients. This can partly be attributed to the use of different analytical methods. In recent years, there has been an increased interest in developing accurate analytical methods valid for the quantification of melatonin in biological samples. Many different analytical approaches, including radioimmunoassay (RIA) [25-301, enzyme immunoassay (EIA) [31,32], gas chromatography-mass spectrometry (GC-MS) [33-371 and liquid chromatography (LC) [38-411, have been reported for the analysis of melatonin in biological systems. Up to now, RIA has been the fastest and simplest technique but, unfortunately, the least selective, owing to its poor reproducibility and its cross-reactivity with other structurally similar compounds. The GC-MS technique is used as a validation procedure. Despite its high selectivity and sensitivity, this technique is not routinely used due to the high cost of the instrumentation. At the present time, LC remains the simplest technique for the determination of melatonin. We report herein an isocratic LC assay with fluorescence detection for melatonin in human serum. Sample clean-up was achieved by “solid phase extraction” with two non-specific serial adsorbents like LC-18 and Carbograph cartridges. This assay is extremely rapid, sensitive and simple. The assay was used to determine serum concentration in eight normal subjects in order to study the circadian rhythm.

2. Experimental 2.1. Chemicals Melatonin (N-acetyl-5methoxytryptamine) and the other standards used were purchased from Sigma (St. Louis, MO). A standard solution was obtained by dissolving a carefully weighed quantity (10 mg) of melatonin in methanol and making up to volume in a 100 ml volumetric flask. This solution was kept at - 20°C. Standard working solutions in water were prepared from the standard solution just before use. Methanol, acetonitrile, dichloromethane and hexane were HPLC-reagent grade (Farmitalia Carlo Erba,

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Milan). The remaining reagents and products were used as received from Farmitalia. Distilled water was passed through a Millipore Milli-Q RG system (Millipore, MA,) 2.2. Other materials The disposable columns used for the sample extraction procedure were Supelclean LC-18 SPE 3 ml tubes (Supelco, Bellefonte, PA,) and 6 cm X 1 cm i.d. polypropylene tubes, filled with 120-400 Carbograph (Altech, Chicago, IL). Prior to use, the LC-18 cartridge was pretreated by percolation of and subsequently with 5 ml of methanol and 5 ml of water. The Carbograph cartridge was pretreated with 5 ml of methanol-dichloromethane (10:90, v/v) and 5 ml of methanol. A Melatonin RIA kit (lz5 I> produced by Buhlmann (Base0 was used to perform the double antibody RIA assay. 2.3. Serum samples Serum specimens were collected from ambulatory healthy donors from “La Sapienza” University Transfusion Center (age 21-55 years), judged to be healthy by review of their medical history, with no evidence of disease. For circadian rhythm studies of melatonin, the sera were obtained from eight volunteer healthy subjects (5 male and 3 female, age 25-44) who did not receive any medication on the days preceding their inclusion in the study. Serum samples were collected at different times over a 24 h period, i.e., at 4.00, 8.00 and 10.00 h a.m.; and 3.00, 6.00 and 10.00 h p.m. Quality controls containing endogenous melatonin were prepared by pooling sera in order to obtain both low and high concentrations of melatonin. The samples were stored deep frozen at -20°C until used for the assay. Specimens were thawed and allowed to reach room temperature before analysis. 2.4. Apparatus The HPLC system consisted of a Binary LC Pump 250 equipped with a Rheodyne Model 7125 injector with 100 ~1 loop (Perkin-Elmer, Norwalk, CT).

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For detection a Model RF-551 fluorescence detector (Shimadzu, Kyoto) (excitation and emission wavelengths of 292 and 345 nm, respectively) was used. The electrochemical detector was a Coulochem II (ESA, Bedford, MA). It included a Model 5021 conditioning cell maintained at a potential of + 100 mV and a Model 5011 dual analytical cell (coulometric-amperometric) set at +350 mV (first cell) and at +500 mV (second cell). Identification and quantification of the melatonin peak was performed using a LCI-100 laboratory computer integrator (Perkin-Elmer). The extraction cartridges were inserted into a “solid phase extraction vacuum manifold system” (Supelco). The note at which samples were applied, rinsed or eluted was adjusted to 2 ml min-’ by means of a slight vacuum. 2.5. Chromatographic conditions A 250 X 4.6 mm column filled with 5 pm (average particle size) Cl8 reversed phase packing and a 20 X 4.6 mm Supelguard LC-18 (5 pm) were used, both from Supelco. The mobile phase for the fluorescence detector consisted of a water-methanol mixture (75:25, v/v); the flow rate was 1 ml min-‘. The mobile phase for electrochemical detection was 50 mM sodium acetate-100 mM acetic acid (pH 4.3), 0.1 mM Na,EDTA, and acetonitrile (75:25, v/v); the flow rate was 1 ml min-‘. All chromatography was performed at room temperature. The concentration of melatonin in the sample was calculated by measuring the peak area of the sample and comparing it with that obtained using a standard melatonin solution. The latter was prepared by drying an appropriate volume of standard working solution and dissolving the residue in 100 ~1 of water-acetonitrile (75:25, v/v). 2.6. Extraction from serum samples

2 ml of serum was passed through an LC-18 cartridge, which was then washed, in turn, with 2 ml of water and 2 ml of water-methanol (90:10, v/v); the washings were discarded. The melatonin was eluted with 2 ml of water-methanol (40:60, v/v).

Chimica Acta 316 (1995) 377-385

This solution was passed through a Carbograph cartridge, and washed with 10 ml of methanol, and then 3 ml of methanol-dichloromethane (80:20, v/v>, which were discarded. Finally, the melatonin from the cartridge was eluted with 1.5 ml of methanol-dichloromethane (10:90, v/v). The eluate was evaporated to dryness on a water bath at 40°C in a flow of nitrogen. The residue was resuspended in 100 ~1 of water-methanol (75:25, v/v) and a 50-~1 portion was injected into the chromatograph.

3. Results and discussion The quantitative assay of a compound, present at a very low level in a complex biological fluid, represents a tremendous challenge in the analysis of a large batch of samples which has to be assayed daily. The determination of melatonin in human serum or plasma is difficult, not because of its low concentration but because of the poor selectivity of the whole procedure (both the LC analysis and the sample extraction). There are only three reports in which melatonin has been assayed in human plasma [39-411. In some studies [39,40] an electrochemical detector was used, while in another [41], the authors utilized a fluorimetric detector. To clean up the human sample, Peniston-Bird et al. [41] used a first extraction step consisting of a solid phase extraction (SPE) using a Sep-Pak cartridge followed by a second purification step involving a double liquid extraction (LLE) with hexane and chloroform, respectively. The LC determination was performed using a water-methanol gradient with a time of analysis of ca. 50 min. An excitation wavelength of 286 nm and an emission wavelength of 352 nm were used for detection. The detection limit of the assay (using 2 ml of plasma sample) was 8 pg ml-‘. The recovery of melatonin in a 2 ml sample of plasma was 70%. The minimal interferences from sample constituents was the priority condition for the assay design for the purpose of establishing the dosage of melatonin in human serum. In this procedure, we introduced a simple selective solid-phase extraction using a two-trap tandem system. A fluorimetric detector was used for quantification in view of its

A. Lagam? et al. /Analytica

reliability. In LC determination the elution of melatonin was achieved in a reasonable time, without any gradient elution, by using a water-acetonitrile isocratic mobile phase with 25% organic modifier. Under this condition, the melatonin was eluted in a retention time of 9.70 min. The resolution of the analyte from other serum components was complete in all the samples studied. 3.1. Choice of detection wavelengths Melatonin was quantitated by fluorescence detection (A,, = 295 nm, A,, = 345 nm), since the maximum fluorescence was observed using these wavelengths unlike those (A,, = 286 nm, A,, = 352 nm> utilized by Peniston-Bird et al. [41]. 3.2. Extraction of melatonin from serum First of all, the serum samples must be carefully purified from most of the endogenous substances likely to cause interference during the LC determination. In an effort to improve the procedure’s selectivity and yield, many parameters were tested, giving the results set below. The extraction of melatonin from plasma in the previously reported procedures involved liquidliquid extraction (LLE) with CH,Cl, [40] or CHCl, [39]. In these cases simple extraction was achieved by means of an enrichment procedure, as described below. The major disadvantage of LLE is emulsion formation, which causes loss of compound and leads to lower, variable recoveries. SPE is generally a rapid and inexpensive method that provides high selectivity and recovery, so we tested it for the extraction of melatonin from human serum. Because non-exchange materials are not applicable to the extraction of melatonin (data no published), a non-specific adsorbent like LC-18 or Carbograph materials was used. Low recovery was obtained for melatonin extracted from a serum sample using only a Carbograph cartridge. This undesirable result may be explained by considering that, as previously reported [42], some unknown carbon-oxygen complexes able to give chemisorption effects are formed on the Carbograph surface in the presence of water

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solutions. Quantitative recoveries are obtained when, after the initial extraction of melatonin by LC-18, it is adsorbed on a Carbograph cartridge from a water-methanol mixture (40:60, v/v). The removal of interfering plasma components is achieved by a washing procedure using solvents of different polarities and several concentration ratios. No difference on the recoveries was observed when the serum was diluted with water before column loading. The concentrations of melatonin in serum and in plasma were compared at the end of the entire extraction procedure. No significant difference in serum or plasma concentrations was observed. Serum was used for the whole study since plasma, when used only occasionally, caused a partial obstruction of the column while percolating through the LC-18 cartridge, thus decreasing the permeability. 3.3. Recovery

and precision

To estimate the recovery of melatonin, a pool of sera was used. Aliquots were spiked with 10, 50, 100 or 200 pg ml-’ melatonin standard solutions. The recovery through the entire procedure was determined by independent determinations, under the same conditions. The recovery efficiency of the assay was determined by subtracting endogenous melatonin from the concentration obtained for supplemented serum. The recoveries of data are summarized in Table 1. The average recovery in the concentration range considered was 88.9% (range 86.3-91.7%). Intra-day precision was established by performing six replicate analyses of a low control concentration and high control sample of known concentrations

Table 1 Results of six replicate serum a Added (pg

ml-‘)

10 50 100 200 a The concentration ml-‘.

analyses

for melatonin

added to pooled

Found (mean f S.D.) (pg ml-‘)

Recovery

20.9 + 1.3 56.4 f 2.9 104.0 f 4.9 191.1 f 7.5

86.3 88.2 91.7 89.4

of melatonin

(o/o)

in the pooled serum was 12.3 pg

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(10.8 and 92.8 pg ml-‘), on the same day. The same low and high samples were also analyzed on five different days to evaluate inter-day precision. Intraday and inter-day coefficients of variation (C.V.) were then determined. The C.V.s for intra-and interassay. variabilities were 7.4 and 8.7% for low serum concentration sample and 5.1 and 7.6% for high serum concentration samples, respectively. 3.4. Linearity and sensitivity The response linearity can be described in terms of the square of the correlation coefficient r. A more exact evaluation provides the variance s2 (residual

Table 2 Potential interferents

in the determination

sum of squares). For the linearity of the detector response, we found the best results with a linear fit in the range from 4 to 250 pg injected for melatonin into the LC system (s2 = 166232, r2 = 0.9991, n = 6). Assuming a signal-to-noise ratio of 2, noise was measured by injecting a blank sample and recording the fluorescence response between 9 and 10.5 min (time interval contained within the retention time of melatonin), the detection limit of the system was 2 pg per injection and the practical limit of the assay for serum was ca. 3 pg ml-’ for 2 ml of sample. An increase in pre-column and column life is achieved using the present procedure. This is due to

of melatonin

Compound

Code

t, (min)

3,4-Dihydroxyphenylethyleneglycol 3-Indolelactic acid 5-Hydroxyindole-3-acetic acid 3-Hydroxyanthranilic aid 3,4_dihydroxyphenylahmine 3-Methoxy-4-hydroxyphenylglycol 3,4_Dihydroxyphenylacetic acid 5-Hydroxytryptophan S-Hydroxyindole-3-acetic acid 5Hydroxytryptophol Vanillylmandelic acid N-acetylserotonin P-hydroxyphenylacetic acid Homovanillyl alcohol Indole-3-acetic acid 6-Hydroxymelatonin Tyrosin Formyl kynurenine N-Acetyltryptophan Tryptophan S-Methoxytryptophan 5-methoxytryptamine Indolelactic acid Tryptamine S-Methoxytryptophol Indoleacetic acid Indole-3-propionic acid Melatonin Tryptophol Indolepropionic acid Epinephrine Dopamine Norepinephrine 5Hydroxytryptamine

DHPG ILA .5-HIAA 3-HA L-DOPA MHPG DOPAC 5-HTRP 5-MIAA 5-HTOL Vh4A NAS HPAA MOPET IAA 6-HMLT TYR FK NATRP TRP 5-MTRP 5MT AIL TAM SMTOL AL4 IPA MLT TOL AIP ADR DA NA 5-HT

1.24 2.67 3.13 3.28 3.58 3.68 3.86 4.13 4.26 4.34 4.57 4.60 4.63 4.77 4.82 4.96 5.05 5.27 5.56 5.93 6.18 6.92 6.95 7.84 8.28 9.07 9.46 9.70 10.81 11.62 > 30 > 30 > 30 > 30

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the fact that we injected a smaller quantity of sample onto the column (50% of the sample) against the 75% [41] and 80% [39,40] reported elsewhere.

3.5. Selectivity Since various serum samples provided different patterns of interfering peaks, the selectivity of the procedure must be investigated. For the detection of melatonin, an electrochemical detector was added next to the fluorimetric detector. The use of a twochannel detector and the possible calculation of peak-area ratios was very advantageous because of increased selectivity, different sera were analyzed only for quantification by the LC system, with the fluorimetric detector and with the electrochemical (EC) detector. The conditions of assay, in the case of the LC-EC, were those reported by Vieira et al. [40]. No significant differences were found when the concentrations of serum melatonin were measured with either type of detector. In order to evaluate potential interferences, 33 compounds (chiefly monoamines) deriving from the metabolism of tyrosine and tryptophan were studied. As can be observed in Table 2, where retention times of single compounds are reported under the same experimental conditions, none of the compounds studied caused interference.

3.6. Comparison

of extraction

b

io

10

TIME (mm) Fig. 2. Chromatogram for a serum sample submitted to our extraction and clean-up procedure. The concentration of melatonin is 39.8 pg ml-‘. See text for chromatographic conditions.

techniques

We compared the procedure of extraction proposed in this work with two extraction procedures in the literature [40,41]. Fig. 2 reports a typical chromatogram relative to the whole procedure of extraction of a serum sample. Figs. 3 and 4 report the chromatograms of the same serum sample utilized in Fig. 2, but with the procedure proposed by Peniston-Bird et al. 1411 (Fig. 3) and Vieira et al. [40] (Fig. 4). As can be noted, in the procedure proposed by Vieira et al. the peak of melatonin is not resolved from other substances. While using the procedure by Peniston-Bird et al., with our experimental conditions, the quantification of melatonin at low concentration would be made difficult by the presence of a peak that eluted near melatonin.

b

io

i0

TIME (mm) Fig. 3. Chromatogram of the same serum sample in Fig. 2 but submitted to the extraction and clean-up procedure proposed by Peniston-Bird et al. [41]. See text for chromatographic conditions.

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0

40

20

60

120

100

80

Melatonin RIA (pg mL-1) Fig. 5. Relation of melatonin RIA for 21 serum samples.

Fig. 4. Chromatogram of the same serum sample of Fig. 2 but submitted to the extraction and clean-up procedure proposed by Vieira et al. [40]. See text for chromatographic conditions.

3.7. Comparison

of LC with RLA

Since most of the laboratories use RIA to detect melatonin, we chose to evaluate our measurements with RIA measurements. We used a commercial kit with [1251] melatonin as tracer and involving extraction with reversed-phase columns for comparative studies with LC. The melatonin concentrations in 21 serum samples measured in the present study were verified by an RIA method (x) and LC method ( y> and the results obtained were analyzed by the least squares methods. The results from all samples assayed are plotted in Fig. 5. There is a close correlation between the concentrations measured in the two methods. The regression equation is y(HPLC) = - 0.6749 + 0.9538x(RlA), r = 0.9842. The range of LC is 6 to 90 pg ml-’ with a mean value of 33.6 f 6.1, pg ml-’ the range of RIA is 5 to 89 pg ml-’ with a mean value of 35.9 f 6.3 pg ml-‘. 3.8. Physiological

studies

The method employed for the determination of melatonin concentration should be of interest for the

concentration

determined

by LC and

large number of samples generated by 24 h studies and yet precise and accurate enough to allow subtle changes in the melatonin rhythm to be detected. The melatonin concentrations of eight normal sub jects (5 female with normal menstrual cycles and 3 male) over a 24 h period are shown in Fig. 6. A typical pattern obtained for the subjects showed lowest concentrations during the day and peak values during the time interval from 10.00 p.m. to 10.00 a.m., with a very high interindividual scatter of

120

0

: , / , / , 13 15 17 IO I

/

/

21

*

,

23

/

/

1

/

/

,

3

,

5

/

)

7

,

L

s

/

,

11

Time of day Fig. 6. Concentration of melatonin in serum samples taken at intervals over 24 h from eight normal subjects (5 female and 3 male).

A. Laganir et al. /Analytica Chimica Acta 316 (1995) 377-385

results. The maximum concentrations of melatonin were obtained around 4.00 a.m. The levels of melatonin that we have obtained clearly showed a circadian pattern and they are in line with the majority of the reported data [5,32,37,40]. It is only by accurate determination of melatonin concentrations that a further insight into the role of melatonin will be achieved. The LC assay described in this paper provides a robust, sensitive, specific and reproducible method for the determination of melatonin. The present method thus proves advantageous compared to the more accurate and extensively used RIA methods, especially in selectivity and reproducibility. In addition, no radioactive reagents are needed and the analysis time is very short. This method should have practical advantages in both clinical and basic studies and can be used also for pharmacokinetic studies when melatonin is used as a drug.

Acknowledgements Financial support of the C.N.R. (National Research Council) and of M.U.R.S.T. (Ministry for the University and for Scientific Research) is gratefully acknowledged.

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