Jul 13, 1984 - sarcosine. We have recently demonstrated (1,2) that a wide range of low-Mi metabolites in urine from humans and animals can be detected ...
CLIN. CHEM. 30/10, 1631-1636 (1984)
UrinaryExcretionof Acetaminophenand Its Metabolitesas Studiedby ProtonNMR Spectroscopy John R. Bales,1 Peter J. Sadler,”3 Jeremy K. Nicholson,2 and John A. Timbrell2 Acetaminophen and its glucuronide, sulfate, N-acetyl-L-cysteinyl, and L-cysteinyl metabolites can be rapidly detected by 1H NMR spectroscopy of intact, untreated human urine. Study of the time course of excretion of these metabolites in five clinically normal men after ingestion of the usual I -g therapeutic dose of the drug showed that the mean 24-h excretion of the drug and these metabolites as determined by NMR was 77.3% of the dose. Respective relative proportions of the above metabolites were 49.9%, 37.6%, 3.0%, and 9.5% (L-cysteinyl plus free drug). Excretion of some other metabolltes in urine, including creatinine, citrate, hippurate, and sarcosine was measured concurrently. Excretion of creatinine and sarcosine was closely correlated. AdditIonal Keyphrases: drug metabolites in urine toxicology detoxificationmechanisms overdosemonitoring urine analysis creatinine citrate hippurate sarcosine .
.
We have recently demonstrated (1,2) that a wide range of
low-Mi metabolites in urine from humans and animals can be detected within a few minutes by high-field proton NMR spectroscopy.The technique is nondestructive and requires no chemical treatment of the sample before analysis. Because initial indications suggested that the technique is also quantitative-estimations of creatinine concentrations by NMR agreed with those by the standard Jaffe method-we embarked on a program to explore the scope of both the qualitative and quantitative use of proton NMR in diagnosis of disease and drug metabolism. There have been two other preliminary reports of studies of urine by proton NMR (3,4). We chose to study the metabolism of acetaminophen (Nacetyl-4-aminophenol, paracetamol) because it is a very widely used analgesic and because several of its metabolites have previously been isolated from physiological fluids and characterized (5-10). Pharmacokinetic studies, by “highperformance” liquid chromatography, have also been reported (5-9). The major metabolites are the glucuronide and sulfate conjugates, but there is considerable interest too in the thiolate conjugates, e.g., with i-cysteine and N-acetyl-icysteine (mercapturic acid) (7-10). These are produced in only small amounts after therapeutic doses of the drug, but become relatively more abundant in casesof acetaminophen overdose and liver damage (7-10). We report here a high-field ‘H NMR study of the urinary excretion of acetaminophen and four of its metabolites, 4glucuronosido-acetanilide, N-acetyl-4-aminophenol sulfate, N-acetyl.2-(i.-cysteinyl)-4-aminophenol, and N-acetyl-2-(Nacetyl-i-cysteinyl)-4-aminophenol (see Table 1), by five clinically normal men over a 24-h period after the ingestion of a 1J)epartment of Chemistry, Birkbeck College, University of London,Malet St., LondonWC1E 7HX, U.K. 2ToxicologyUnit, Department of Pharmacology,Schoolof Pharmacy, University of London, Brunswick Square, London WC1N lAX, U.K. 3To whom correspondenceshouldbe addressed. Received May 16, 1984; accepted July 13, 1984.
single therapeutic dose (1 g) of the drug. We have also characterized several other possible metabolites by ‘H NMR spectroscopy. Excretion of some other urinary metabolites (creatinine, citrate, sarcosine, and hippurate) was also monitored siniultaneously by ‘H NMR spectroscopy. The results demonstrate the potential of NMR in the study of the broader picture of metabolic changes induced by drug administration. typical
MaterIals and Methods We studied the excretion of acetaniinophen and its metabolites by five healthy men, ages 23-37 years, whose weights fell within a narrow range (67-73 kg). Each ingested two 500-mg (6.62-mmol) tablets of acetaminophen (Paracetamol BP), equivalent to a dose of between 91 and 99 p.mol of drug per kilogram of body weight. One subject repeated this experiment. The drug was taken at mid-morning, immediately after a control urine sample had been collected. During the experimental period, subjects ate and drank without restriction and no other drugs or alcohol was taken. Urine specimens were collected at hourly intervals up to 6 h, and then the total urine from 6 to 24 h was pooled. The volume and pH (all between 5.3 and 7.4) of each sample were recorded. Samples were frozen immediately and stored at -20 #{176}C until analysis. We transferred 0.45 mL of urine, after thawing, to a 5-mm NMR tube and added 50 L of 21120 to each to provide an internal field-frequency lock for the spectrometer. To quantify the urinary metabolites, we added 50 L of a 200 mmol/L solution of L-valine in 21120 to 0.45 mL of urine to give a final valine concentration of 20 mmol/L. We made this addition either to the complete series of urines from one subject, or to one sample of a series, all of which we analyzed by ‘H NMR, using identical spectrometer and processing conditions. Concentrations were determined by comparing either computer-integrated peak areas or peak heights of the urinary metabolite resonances with those of added valine. Given sufficient digital resolution in the spectrum and constant line-widths, there was a reasonable correspondence between the ratios of peak heights and areas. To test analytical recovery, we recorded 1H NMR spectra of five identical urine samples after the addition of an equal volume of an 2H20 solution containing acetaminophen; its glucuronide, sulfate, and N-acetyl-i-cysteinyl conjugates; and 3-(trimethylsilyl)[2H]4propanoic acid (TSP) for reference (at 2.9, 4.9, 5.7, 3.1, and 2.5 mmol(L, respectively). Spectra were recorded under identical spectrometer conditions. We usedBruker Model AM500 (Biomedical NMR Centre, Mill Hill) and WH400 (University of London facility, Queen Mary College) NMR spectrometers with a Bruker Aspect 2000 data system. All spectra were recorded at ambient probe temperature (25 ± 1 #{176}C). For each sample, we accumulated 48 free-induction decays (F1Ds), using 16 384 data points, a 45600 pulse, and about a 5-s pulse repetition rate; when we used a 10-s pulse repetition rate, there was no CLINICALCHEMISTRY,Vol. 30, No. 10, 1984
1631
no substantial
change in the relative peak intensities, which suggests that spin-lattice relaxation was complete within 5s. The intense water resonance was suppressed by applying either gated (with the decoupler switched off during acquisition) or continuous secondary irradiation. None of the metabolites quantified in this paper have resonances that closely overlap with the H20 signal. FIDs were zero-ifiled to 32 768 data points before Fourier transformation to increase digital resolution, and an exponential function corresponding to a 1-Hz line broadening was applied. The spectra were referenced to either the CH2 singlet resonance of glycine (3.57 ppm) or the aromatic doublet resonance of hippurate (7.83 ppm), which do not shift in the normal pH range of urine (1). ‘H NMR spectra of several potential metabolites were recorded in deuterated phosphate buffer (0.1 mol/L, pH” 5.8), referenced by addition of [2H]4TSP (#{244} = 0 ppm).4 Table 1 gives structures, chemical shifts, and coupling constants. For acetaminophen, its glucuronide, sulfate, and N-acetyl-icysteinyl conjugates, we monitored a pH titration with NMR. Over the normal pH 5 to 8 range of urine there were
shifts (>0.02 ppm) in the resonances of these
compounds.
Results and Discussion The main problem in recording ‘H NMR spectra of intact urine is the large resonance from H20. However, this can be adequately overcome by applying gated or continuous secondary irradiation at the H20 frequency (1). Peaks from acetaminophen and its major metabolites are then readily detected in spectra of urine excreted several hours after ingestion of the drug. Figure 1 depicts a typical 500-MHz ‘H NMR spectrum obtained from urine collected 6 h after drug ingestion, and it serves for discussion of peak assignments. These were aided by first recording and assigning peaks for acetaminophen itself and those of potential metabolites (Table 1). A major advantage of NMR is that “fingerprints” of molecules often consist of more than one resonance, each with characteristic shifts and intensity ratios, and sometimes exhibit additional spin-spin coupling. In favorable cases this allows unambiguous identification of the substance. (In contrast, traces for liquid-chromatographic effluents, obtained with electronic
pH, the pH-meter reading in 2H,O.
n
G
n
G Formate
NAC A
I-il’ D
CreatinineI CH3
Dihydroxy
2:20
acetone
2.15
2#{149}20 215
Sarcosine
NAC
I
I
4
3
2
&/ppm
Fig. 1.500-MHz 1HNMR spectrumof a urine samplecollected6 h afteracetaminopheningestion,showing thearomatic (tippet) and aliphatic (k,we,) regions Conditions as describedin text exceptforinsertb, wheregaussianresolutionenhancementwas used.A, acetaminophen; G,gkjcuronide;S.sulfate;C, cysteinyl;N4C, acetyl-tcysteinyl derivativeswith assignmentsas in Table 1. Assignmentsof other peaks areasreportedin ref. I with the additionof hippurateNH at 8.54ppmand sarcosineN-CH3at 2.75 ppm 1632 CLINICAL CHEMISTRY, Vol. 30, No. 10, 1984
Table 1. ‘H NMR Chemical Shifts (&tppm) and Coupling Constants (J/Hz) for Acetaminophen and Some Possible Metabolites5 Coupling constant, Chemical Name
R group
Phenolicconjugates
H1
shift, &‘ppm
H
H,,
J/Hz
A
Hd
(H.-HJ
(H1-H,,)
HNCOCH aa DUb
OR Acetaminophen
-H
7.25(d)
6.90(d)
2.15(s)
8.7
Sulfate
-SO3K
7.45(d)
7.31(d)
2.17(s)
8.9
7.34(d)
7.13(d)
2.16(S)
7.23(dd)
6.93(d)
7.42(d)
CO2H
Glucuronide H
5.11 (d), 3.60-3.94
8.9
OHA OH OH
H
3-Positionconjugates HNCOCH
bR
NAcetyI-L-cysteinyl
2.14(s)
1.84(s), 3.28(ABX), 8.7
-S-CH2-CH-NH-CO-CH3
2.1
4.30(X) CO2H
-S-CH2-CH-NH2
i-Cysteinyl
3.99(X)
8.7
7.26(dd)
6.99(d)
7.51(d)
2.15(s)
3.35(ABX),
7.41(dd)
6.99(d)
7.61(d)
2.17(s)
2.93(s)
8.7
2.4
CO2H OH
Methylsulfinyl
-S-CH3 0 Thiomethyl
-S-CH3
7.08(dd)
6.91(d)
7.30(d)
2.15(s)
2.44(s)
8.6
2.2
Methoxy
-O-CH3
6.83(dd)
6.90(d)
7.08(d)
2.15(s)
3.85(s)
8.4
1.9
Glutathionyl
-Cys
7.22(dd)
6.94(d)
7.45(d)
2.15(s)
2.08(q), 2.43(dq)
8.7
2.3
8.6
2.50
GIu -
Gly
3.31 (ABX), 3.62(AB)
3.73(t), 4.46(X) 2-Positionconjugates
HNCOCHg aR
bL)c OH Glu
Glutathionyl
-Cys ((
7.19(d)
6.83(dd)
7.05(d)
2.19(s)
Gly
2.08(q), 2.41(dq) 3.34(ABX), 3.66(AB)
3.73(t), 4.51(X) 1AlI solutionswereabout10 mrnol/L, in deuterated phosphate buffer. pH 5.8. bHbHc =
doublet of
quartets,(AB)
=
second-order AB multiplet, (ABX)
=
AS part of ABX, (X)
absorption or fluorescence detectors, give only a single peak for each metabolite.) The acetanilide N-acetyl groups of the metabolites all gave sharp signals in the rather narrow range from 2.13 to 2.19 ppm. These were well-resolved at
(a) singlet, (d) =
=
X
=
doublet, (dd) = doublet of doublets, (q)
=
quartet,(dq)
part of ABX.
400 or 500 MHz if the resolution was enhanced by application of a gaussian function to the FID before Fourier transformation (Figure 1). Fortunately, this region is relatively clear in spectra of urine from normal human subjects. CLINICALCHEMISTRY,Vol. 30, No. 10, 1984 1633
of each metabolite, based on the relative peak heights of the individual N-acetyl resonances. The addition of a standard solutionto five replicate urine samples showed that the analytical recovery of total acetaininophen plus metabolites was good (97.7 ± 2.4%, mean ± SEM). The calculation of the relative proportions of each metabolite is subject to an error (of up to 10% on individual samples)related to partial overlap of resonances;however,
The only interference we have detected so far is from Nacetylcarrntine in urine from subjects who had fasted for more than 24 h. Acet.aminophenmetabolites have other characteristic ‘H NMR resonances (Fable 1). In addition to the N-acetyl resonances, the aromatic ring protons give rise to reso-
proportion
nances with clearlyidentifiable coupling patterns, spread over about 0.6 ppm (Figure 1, Table 1). In the case of the glucuronide, the f3-anomeric proton of the sugar-ring has a further characteristic doublet resonance at 5.11 ppm (J = 6.0 Hz) and also resonances between 3.6 and 3.9 ppm, corresponding to other ring-protons. An additional assignment aid for the 3-(N-acetyl-i-cysteinyl) conjugate was the sharp singlet for the side-chain N-acetyl group at 1.84 ppm. This analysis made it clear that ‘H NMR might be used to study quantitatively the urinary excretion of acetaminophen and its major metabolites the glucurothde, sulfate, Nacetyl-icysteinyl, and i-cysteinyl conjugates.Other possible metabolites (see Table 1) were not detected in the urine samples studied here. The above-described urine samples from one of the five men showedthe time course for the excretionof metabolites illustrated in Figure 2, for the region from 1.8 to 2.8 ppm. In addition to the N-acetyl peaks for acetaminophen metabolites, other signals from normal urinary metabolites were observed in this region-for example, a distinctive quartet for the CH2 protons of citrate (an AB spin system), which shifts with pH (1), and a nearby singlet (2.75 ppm), which we have now assigned to the N-CH, protons of sarcosine (Nmethyl-i1-glycine). The latter is a useful reference for alignment of peaks, because it does not shift within the usual pH range of urine. To quantify the metabolites, we first measured the total peak area of the acetanilide N-acetyl resonances relative to the methyl doubletsof valine (at 1.0 ppm) added as a concentration standard. We then calculated the
this can be reduced by resolution-enhancement techniques (for example, applying a gaussian function to the FID). In view of the overlapbetween the N-acetyl signals of the free drug and the Ircysteinyl conjugate,we estimated their combinedvalues. Figure 3 shows,for our six experiments, the mean excretion of the glucuronide, sulfate, N-acetyl-icysteinyl, and the sum of free drug and i..-cysteinyl conjugate during the 24-h period. The data are plotted as a percentage ofthe total concentration of all observed metabolites at each time point. The glucuronide:sulfate ratio is initially
