Identification and quantitative determination of saccharin in biological fluids

BIOCHEMICAL

MEDICINE

Identification

8,

and

362-370

( 1973)

Quantitative

Determination

in Biological M. W. COUCH, Veterans

Fluids

N. P. DAS, K. N. SCOTT, Administration University

Hospital

of Florida Gainesville,

of Saccharin

AND

C. M. WILLIAMS

and Department College of Medicine, Florida 32601

of Radiology,

AND

R. L. FOLTZ Battelle

Received

Columbus Columbus,

Laboraton’es, Ohio

November

20, 1972

The determination of saccharin in biological fluids has in the past been carried out by paper and thin-layer chromatography (l-5), ion exchange chromatography ( 6), infrared spectrometry ( 7)) and ultraviolet spectrometry (S), but no techniques for its determination by gas-liquid chromatography (glc) have been described to our knowledge. We have been engaged in the identification of low molecular weight constituents of urine and cerebrospinal fluid, and this paper concerns the identification of saccharin as its N-methyl derivative in urine and plasma and provides a simple method for its quantitative determination by gas chromatography ( gc). The identification procedure involved the isolation of the compounds from urine and plasma by preparative gc, followed by preliminary characterization by high and low resolution mass spectrometry. Further confirmation of the structure of the compounds was accomplished through ‘H (PMR) and ‘“C (CMR j nuclear magnetic resonance spectroscopy of the urinary compounds, which can generally be obtained in larger quantity than the compounds from the plasma. Once the structure of the compounds was established by mass and nuclear magnetic resonance (NMR) spectroscopy, final proof of the structure was obtained by comparison with the mass and NMR spectra and gc characteristics of the authentic compound. Copyright AH rights

0 1973 by Academic Press, of reproduction in any form

362 Inc. reserved.

SACCHARIN

DETERMINATION

IN

BIOLOGICAL

FLUIDS

36>3

EXPERIMENTAL

Analytical gc was carried out on a F & M Model 400 gas chromatograph using a 6 ft X 3 mm i.d. glass column with 3% OV-17 on 80/100 Chromosorb G; injector, 270°C; flame ionization detector (HZ), 330°C; column loo”-300°C at 3’C/min; carrier gas (N,), 60 ml/min. Relative retention times were expressed in terms of Kovats Indices (KI) (9). A Varian 90-P3 with thermal conductivity detector was employed for preparative gc using a 5 ft x 5 mm i.d. stainless-steel column with 3% OV-1’7 on SO/l00 Chromosorb G; injector, 270°C; detector, 300°C; collector, 270°C; column, lOO”-300°C at B”/min; carrier gas (He), 60 ml/ min. Low resolution electron impact mass spectrometry ( EIMS ) was carried out on a CEC 491 mass spectrometer, the transitions leading to metastables being determined from ion kinetic energy (IKE) measurements ( 10). An AEI MS-9 was employed for high resolution EIMS and was operated at an effective resolving power of 15,000. Proton and ‘“C NMR spectra were obtained on a Bruker HX-90 spectrometer equipped with a fast Fourier transform system using a Nicolet 1083 computer. For urinary N-methyl saccharin, Fourier transform NMR spectra were obtained of a 0.2 ml deuteriochloroform solution, which contained 5% by volume hexafluorobenzene as the frequency stabilizing lock material and 2% by volume tetramethylsilane (TMS ) as the chemical shift reference material. A 5 mm diam sample cell with a vortex plug was employed. The 13C spectra obtained were broad-band proton noise decoupled to yield good signal-to-noise ratio. In order to obtain gas chromatograms, the following extraction procedure was followed. Twenty milliliters of urine was acidified to about pH 1 with 6 M HCl, the solution saturated with salt and extracted with three “O-ml portions of ethyl acetate. The ethyl acetate was removed by a rotary evaporator in vucuo. Excess ethereal diazomethane (11) was added to the residue and allowed to react for about 25 min (until nitrogen evolution ceased) before evaporation of the solvent in VUCUO.One milliliter of plasma samples were acidified to about pH 1 with 6~ HCl and extracted with three 2-ml portions of ethyl acetate. On evaporation of the solvent the residue was treated as described above. Saccharin was prepared by dissolving sodium saccharin (Peni& reagent grade) in water and adding excess 6 M HCI. The precipitate was extracted with ethyl acetate and crystallized by evaporating the solvent. N-Methyl saccharin was synthesized by treating saccharin with excess ethereal diazomethane. Benzene sulfonamide was obtained from Aldrich Chemical CO. The four sulfanilamides investigated by us were obtained commercially: sulfacetamide and sulfamerazine from Mann Chemicals.

364

COUCH

FIG.

1. Gas chromatogram

(OV-17)

ET AL.

of a methylated

acid

urine

extract.

sulfonilamide from Penick, and sulfadiazine from Lederle Laboratories. These sulfanilamides were treated with excess diazomethane to give compounds assumed to be their N-methylated derivatives. RESULTS

AND

DISCUSSION

The chromatogram of a methylated urine extract from a mentally retarded child (Fig. 1) showed several large peaks. The compound appeargc and shown to give ing at KI 1995 (I) was trapped by preparative a single peak at KI 1550 on 3% SE-30. Mass Spectrometry The intense molecular ion observed at m/e 197 in the low resolution EIMS of KI 1995 was shown, by high resolution EIMS, to have the empirical formula C,H,NO,S (calcd for C,H,NO,S: m/e 197.0147. Found: m/e 197.0160). The major fragment ions suggested that I was the Nmethyl derivative of saccharin and its mass spectrum was found to be identical, within experimental error, to the spectrum of authentic Nmethyl saccharin reported by Hettler et a?. ( 12). Our mass spectrum of I (Fig. 2) shows the hitherto undetermined empirical formulas of the major fragment ions. High resolution EIMS and IKE measurements further clarified the fragmentations of N-methyl saccharin (Fig. 3 ) Nuclear

Magnetic

Resonance

Spectroometry

Ten milligrams of the compound appearing at KI 1995 (I) was trapped by preparative gc and examined by PMR and CMR. Allowing for the difference in concentrations, the ‘H and 13C spectra of urinary

SACCHARIN

DETERMINATIOS

IN

BIOLO(;ICAL

FLUIDS

m/e

FIG.

2. Mass

spectrum

of N-methylsaccharin.

N-methyl saccharin were the same as the spectra of authentic N-methyl saccharin in saturated deuteriochloroform solution. The aromatic region of the proton spectrum of N-methyl saccharin is a complex multiplet covering the 8.14-7.79 ppm range. The necessary computer analysis to

-co -YN = CH2

---I

+

C&NO

1 + 133 L-

m/e

-co

C6”4 132 i

C7”40 m/e 104

4 I

C,H6N m/e

CT”~~ m/e

.

4 104

105

++L%“5 cio 1 D 1 m/e 1

-

CH2N

* -CH2N

0

77

mle

FIG.

105

3. Fragmentation

pattern

of N-methylsaccharin.

m/e

76

366

Uetlzene N-Methyl

saccharin”

sulfonamided

Sac:charinc ----.-~

CartJoIle

c=o cs C6 C5 C9 C4 c7 NCH,

Chemical 159.1 138.3 134.9 134.6 130.9 125.5 121.4 23.3

shift

Carbona c=o C8 C6 c5 c9 c4 c7

or 134.6 or 134.9

Chemical 163.3 141.2 137.4 136.7 129.5 126.8 123.0

shift

or 136.7 or 137.4

Carbon CHO. 2 C para C meta C ortho

(1 In ppm from TMS. * Ten milligrams of urinary N-methyl saccharin in 0.2 ml deuteriochloroform hexafluorobenxene and 2y0 TMS. c Sat$urated solution of authent,ic saccharin in perdeuteriodimethyl sulfoxide

(Ihemical shift 14,5.7 133.5 130.5 127.3

with

5%

with

570

hexafluoroacetone. d Saturated ide

solution

of authentic

benzene

sulfonamide

in perdeuteriodimethyl

sulfox-

with 5(1, hexafluorobenzene. e Numbering

of aromatic

carbons

as in Fig.

4.

extract chemical shifts for the aromatic protons was not performed. The proton spectrum also contains the NCH, peak at 3.28 ppm. The “‘C chemical shifts are summarized in Table 1. The chemical shifts of the carbons in the aromatic region of the spectrum were assigned by assuming additivity of substituent effects on the chemical shift (13). The ‘6 chemical shifts of methyl hippurate (14) and of benzene sulfonamil’de (Table 1) were used to obtain the necessary substituent effects. Figure 4 shows the ‘:‘C spectrum of a saturated solution of authentic saccharin in perdeuteriodimethyl sulfoxide. For this more concentrated solution, a ‘“C spectrum without proton decoupling could be obtained, and the peaks assigned to carbons 4-7 are seen to be doublets owing to coupling with directly attached protons. A search of the literature revealed no previously reported PMR or CMR data for saccharin or N-methyl saccharin. Determination

of Saccharin

in Urine and Plasma

Sodium saccharin (120 mg) was administered orally to a normal subject, lirine collections were carried out over the subsequent O-6, 6-12, and 12-24 hr periods. Blood samples ( 4 ml) were obtained from the subject’s arm vein 5 miu before oral intake of sodium saccharin and at 6, 12, and 24 hr. Each blood sample was collected in a glass centrifuge tube containing a few crystals of sodium oxalate and was centrifuged to

SACCHARIN

DETERMINATION

IN BIOLOGICAL

FLUIDS

FIG. 4. 22.63 MHz T NMR spectrum of authentic saccharin with 5% hexafluoroacetone deuteriodimethyl sulfoxide solution, stabilizing lock material. Spectral width: 5000 Hz; upper trace; spectrum; lower trace: normal spectrum.

36’i

in saturated peras spectrometerproton decoupled

obtain the plasma. Aliquots of the urine and plasma extracts were treated with diazomethane as described in the experimental section. In order to establish that saccharin is not methylated in viuo, another sample of urine containing saccharin metabolites was extracted by the usual procedure but was not treated with diazomethane. A gas chromatogram of this extract showed no traces of a peak at KI 1995, indicating that methylation of saccharin is not a metabolic route in man. An earlier report of saccharin metabolism in rhesus monkeys showed that saccharin was largely excreted unchanged. Only trace amounts of two metabolites, o-sulfamoylbenzoic acid and ammonium-o-sulfamoylbenzoic acid were present ( 1) . The peak corresponding to N-methy saccharin was detected in all the urine samples but was present only in the 6 and 12 hr plasma samples. The total amounts of saccharin in the urine and plasma were determined by gc using nonadecane (Aldrich Chemical Co., Inc.) as an internal standard. The results are presented in Table 2. A straight-line

368

COUCH

ET AL.

TABI,E

2

ESTIMATION OF N-IMETHYLSACCHARIN IN URINE AND PLASMA AFTEX THF, ADMINISTRATION OF SODIUM SKCHARIN (120mg) TO MAN Time -

(hr) --__-__

-.__

Urine

(mg)

6 12 24 Total

% Dose

52 13 50

in urine

Plasma

43 11 42

iii

(mg/lOO

ml‘1

8.1 2.2 0

96

correlation was observed (Fig. 5) for the amount of N-methyl saccharin (in the range of 1 to 6 pg) vs the peak area ratio (N-methyl saccharin/ nonadecane ) as determined by gc. Quantities as small as 120 ng could be detected in a standard solution of N-methyl saccharin. However, in a trypical urine extract, the close proximity of a large methyl hippurate peak (KI 2035 + 20) limited the detection of N-methyl saccharin to about 250 ng. In the first 24 hr, 96% of the administered saccharin was excreted unchanged in the urine. Similar results were obtained by Pitkin et al. (1) for saccharin metabolism in monkeys. In our study, about 43% of the administered saccharin dose was excreted 6 hr after oral intake of the compound. The percent recovery of saccharin using ethyl acetate extrac-

Conccnir~lion

of

N-methylsaccharin

Cpg)

FIG. 5. Standard curve of N-methyl saccharin using nonadecane standard. (Each point on the curve is an average of three determinations, line representing the range of values obtained.)

as internal the vertical

SACCHARIN

DETERMINATION

IN

BIOLOGICAL

FLUIDS

369

tions on five urine samples in duplicate containing different amounts of saccharin was 98.5% ( & 1.7 SD). In order to determine whether saccharin is bound to proteins in the blood, 1 ml of trichloroacetic acid (15% w/v) was added to a l-ml aliquot of each plasma sample. The resulting mixtures were centrifuged and the plasma proteins separated from the supernatant. Both the plasma proteins and supernatant were extracted with ethyl acetate (3 X 2 ml), derivatized with diazomethane and analyzed by gc. N-Methyl saccharin was present in the plasma protein extracts of the 6 and 12 br collections, but was absent in all the supernatant (plasma protein free) extracts. This suggests that, in blood, saccharin occurs bound to the plasma proteins and may be extracted from the proteins with ethyl acetate. Sulfonamide drugs in current use were found not to interfere with the determination of saccharin. Without derivatization, all these sulfonamides, as well as saccharin itself, exhibited broad tailing peaks on an OV-17 column. On treatment with diazomethane each sulfonamide gave a single symmetrical peak at a different KI than N-methylsaccharin. Presumably, diazomethane methylated the nitrogen adjacent to the SO, portion of these compounds; however, no investigations have been undertaken to confirm this supposition. None of the sulfonamides are bicyclic as is saccharin. Methylated sulfanilamide and sulfacetamide, the sulfonamides most cIosely resembling N-methyIsaccharin in structure. appear at KI 2630 and 2655, respectively, on OV-17. Sulfadiazine and sulfamerazine appear at KI 3305 and 3320 on OV-17, respectively, after methylation. SUMMARY

A gas chromatographic method for the qualitative and quantitative determination of urinary and plasma saccharin as its N-methyl derivative has been described. The structure of this derivative was proved by mass spectrometry and by proton and 13C NMR. About 96% of the saccharin was excreted unchanged in the first 24 hr urine collection after oral intake of saccharin by man. This method is relatively simple and rapid and may be adapted for other biological fluids. ACKNOWLEDGMENTS Financial support by the Veterans Administration and the National Institutes Health (Grants NO. GM-16788 and No. NS-09576 and Contract No. 69-2226) the China Med’cal Board, New York (Dr. Das) is gratefully acknowledged.

of and

REFERENCES

1. PITKINS,

M., ANDERSON, D. W., REYNOLDS, W. A., AND FILER, L. J., JR., Proc. Sot. Biol. Med. 137, 803 (1971). 2. ICRIBAGASE, H., AND KOJIMA, S., Yakuguku Zasshi 82, 1616 (1962); Chem. Ah&. 58,

R. Exp.

8357g.

370

COUCH ET AL.

3. SALO, T., AND SALMINEN, K., Suomen Kemistiiehti (Pt. A. ) 37, 161 (1964); Chem. Abstr. 62, 3382f ( 196.5). 4. KOJIMA, S., AND ICHIBAGASE, H., Yakuzaigaku 2966, 115; Chem. Astr. 70, 2486s (1969). 5. WOIDICH, H., GNAUER, H., AND GALINOVSKY, E., Z. Lebensm. -Unters. Forsch 133, 317 ( 1967); Chem. Abstr. 69, 52795r ( 1968). 6. ASANO, K., TAIRA, M., NAKANISHI, H., SENDA, E., SHIRAISHI, Y., AND TAKESHITA, R., Nuchidai Igaku Zarsshi 22, 797 (1963); Chem. Abstr. 61, 8813h (1964). 7. NAGASE, Y., BARA, S., AND SUZUKI, M., Yakugaku Zasshi 79, 705 (1959); Chem. Abstr. 53, 19300a (1959). 8. BRADFORD, L. W., AND BRACKETT, J. W., Mikrochim. Acta 353 (1958). 9. sz KOVATS, E., Advan. Chromutog. 1, 229 (1965). 10. KISER, R. W., SULLIVAN, R. E., AND LUPIN, M. S., Anal. C&m. 41, 1958 (1967). 11. MCKAY, A. F., J. Amer. Chem. Sot. 70, 1974 (1948). 12. HEALER, H., SCHIEBEL, H. M., AND BUDZIKIEWICZ, H., Org. Mass Spectrom. 2, 1117 (1969). 13. SCOTT, K. N., J. Amer. Chem. Sot. 94, 8564 ( 1972). 14. COUCH, M. W., GREER, M., SCOTT, K. N., AND WILLIAMS, C. M., J. Neurochem. 20, 893 ( 1973).