2580
Anal. Chem. 1985, 57,2580-2583
Detection of Thioethers of Pharmaceutical Importance by Liquid Chromatography with On-Line Generated Bromine W. Th. Kok, J. J. Halvax, W. H. Voogt, U. A. Th. Brinkman, and R. W. Frei* Department of Analytical Chemistry, Free University, De 1!3oelelaan 1083, 1081 HV Amsterdam, T h e Netherlands
A new method is presented for the detectlon of thloethers after separatlon by high-performance llquld chromatography (HPLC). The method Is based on the postcolumn oxldatlon of the thloethers by bromine, which Is generated electrochemically In the column effluent. Detectlon Is performed by amperometrlc measurement of the excess of bromine downstream, after reactlon wlth eluting thioethers. For the determlnatlon of amplclllln In plasma and urlne a superlor sensltivlty and selectlvlty compared to the usual UV dstectlon Is shown. Plasma concentratlons from 0.2 to 10 pg/mL can be determlned, with a preclslon of &e%. Attentlon has been paid to the kinetics of the reaction of bromlne wlth various penlcllllns and thelr degradation products. Ranitidine can be determlned In plasma after sample cleanup and concentration by Ilquld-liquid extractlon wlth a lower llmlt of detection of 2 ng/mL. On the other hand, a much better reproduclble method was developed wlthout extraction steps, wlth whlch ranltidlne levels from 10 to 300 ng/mL can be measured, wtlh a preclslon of & I O % In the 100-300 ng/mL range. With thls procedure an Internal standard does not have to be used.
While a number of specific detection techniques have been developed for the determination of thiols and disulfides after separation by HPLC, no general method is known for organic compounds with thioether groups. Amperometric detection with mercury (film) electrodes, introduced by Rabenstein and Saetre (I) for thiols and also applied to thioketones (2),is not possible for thioethers since mercury compounds are not readily formed. With other electrodes oxidation potentials are high ( 3 ) and electrode passivation and adsorption phenomena serious ( 4 ) . Methionine for instance can only be oxidized a t a useful electrode potential in aqueous soltions when it is adsorbed on a metal oxide which acts as a catalyst (5-7). Also with other detection systems, such as a ligandexchange method @ ) , the reactivity of thioethers is low compared to thiols. Therefore, when no other reactive groups are present in the molecule, thioethers me now generally detected by UV absorbance measurements. Contrary to the troublesome electrochemical process, the homogeneous reaction of thioethers with a variety of oxidants is often fast and straightforward, the products in aqueous solutions formed being mostly sulfoxides or sulfones depending on the reaction conditions (9,lO). We have used the homogeneous oxidation of thioethers by bromine for a specific detection technique, which is shown in a number of cases to be more sensitive and more selective than UV absorbance detection. The HPLC detection method involves the electrochemical generation of bromine in the column effluent from bromide present in the mobile phase. The bromine can react with compounds eluting from the column in a reaction capillary, and compounds of interest can be quantitated by amperometric measurement of the excess of bromine downstream. Figure 1 gives a schematic impression of this method. It has been used before, for the determination of prostaglandins (11)
and opiates (12). As was shown in previous work (12),with careful optimization of the reaction time and generation current, low detection limits can be obtained despite the fact that the signals obtained are basically current differences. Since the bromine reagent is produced on-line, mixing problems are absent and noise levels can be kept low. One example studied in detail is the determination of ampicillin (I), an often prescribed antibioticum, in body fluids. LOOH
I
The drawbacks of the method now generally used, HPIX with UV detection a t about 220 nm (13, 14), are the limited selectivity and sensitivity. Detection limits reported are in the Irg/mL range for plasma samples. Both precolumn (15, 16) and postcolumn (17-20) derivatization techniques have been proposed to improve the detection of ampicillin or related compounds. Detection limits after derivatization range from 0.1 to 1pg/mL for plasma. Only with laborious sample extraction procedures (16,18)can the sensitivity be improved further. In this work we have compared the performance of our detection system with UV detection for ampicillin in plasma and urine. Another application studied is the determination of' ranitidine (11) in plasma. Ranitidine is a drug meant to replace (CH3)2- N - CH2 e C t i 2 - S-Cti2CH2-NH-C-NNH--CH3 Ii HC - NO2
11
cimetidine in ulcer treatment. It is now determined in plasma by HPLC with UV detection after one or more extraction steps (21-23). Amperometric detection with plasma extracts is also possible but suffers from electrode passivation phenomena (24). Reported extraction efficiencies range from 47 to 8470, and internal standards have to be used. Lower limits of detection from 5 to 20 ng/mL plasma are given in the literature. We have compared the sensitivity of our method for ranitidine in plasma extracts with these literature values. Moreoever, we have developed a method which eliminates the need for the cumbersome extraction steps.
EXPERIMENTAL SECTION Equipment. The chromatographic equipment consisted of a Perkin-Elmer 601 pump which, unless stated otherwise, was set up to a flow rate of 1 mL/min, a Rheodyne injection valve with 20- and 1OO-wL loops, and 4.6 mm i.d. columns home-packed with LiChrosorb bonded phase materials. UV detection was performed with a Pye Unicam LC 3 variable wavelength detector. The cell used for the generation of bromine (which we have named the
KOBRA cell) has a double thin-layer construction, with the electrode compartments separated by an ion-exchange membrane. Its basic concept and performance characteristics have been
0003-2700/85/0357-2580$01.50/00 1985 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 13, NOVEMBER 1985
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9-
_------ I Ir- i
3t
10 -tr[s]
20
@
' t //
kd
0 0
100 -a..
IG
[la]
200
Flgure 2. Number of bromine molecules reacting with one molecule of ampicillin (0)or ampicilloic acid ( 0 )with different reaction times (a) or generating currents (b): (a) 1, = 50 PA; (b) t , = 5 s.
Table I. Recovery of Ampicillin from Plasma after Deproteinization ampicillin added. &/mL 0 2 4 6 8 10
ampicillin found,np g K serum A serum B 0.00 f 0.00 1.56 f 0.06 2.92 f 0.32 4.50 f 0.36 6.00 f 0.32 7.30 f 0.40
mean
recovery, %
0.00 f 0.00 1.58 f 0.04 3.00 f 0.12 4.56 f 0.30 6.24 f 0.00 8.00 f 0.20
78
74 75 76
79
Mean f standard deviation from triplicate injections. with more than one molecule of bromine, but the kinetics are much slower. From the curves in Figure 2 at low generation currents and short reaction times, the second-order rate constant k 2 of the first oxidation step can be evaluated. Calculation yielded
kz = 1.3 X lo4 L mol-'
ssl
Previously (12) it was shown that the lowest detectable analyte concentration is equal to 2d,/k2tr, where d, is the relative level of the noise on the base-line signal and t, is the reaction time. For our system, with d, = 0.002 and t, = 12 8, the lowest detectable peak concentration of ampicillin will be 2.6 x M, which corresponds to a sample concentration of 0.1 pg/mL. This limit of detection will be obtained with generating currents below 20 FA. To obtain a wider linear range we chose a somewhat higher generating current, 50 pA, for the analysis of body fluids. A linear range of 0.2-10 pg/mL is predicted. With standard solutions no deviations from linearity were found experimentally within this range. Peak height reproducibility was within f2% for standard solutions on the 2-10 pg/mL level. Chromatography. The recovery of ampicillin from plasma after deproteination was studied by using two pooled sera from different sources, spiked with various amounts of ampicillin. Peak heights obtained were compared with those of standard solutions. The results are given in Table I. The recovery from all samples examined was 77 f 5% (mean standard deviation, n = 10). The recovery percentage is not depending significantly on the ampicillin concentration or the plasma used.
*
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 13, NOVEMBER 1985
0
5
0 10
0
5
10
15
t[min]
i[rnin]
+
Figure 3. Chromatograms obtained with deproteinized plasma: (a)
-
5
I
_
10 t [min]
_
0
5
10
t[min]
Flgure 5. Chromatograms obtained with plasma extracts: (a) blank plasma; (b) plasma spiked with ranitidine (22 ng/mL).
blank plasma; (b) plasma spiked wlth ampicillin (8 pg/mL).
1 0
-
10
5
t[rnin]
15 0
IrnAUI
--. 5
,I
10 t [rnin]
15
Flgure 4. Chromatograms obtained with urine, splked wlth ampicillin (100 pg/mL), diluted 1: 10: (a) bromine-generation detection system; (b) UV detection.
Figure 3 shows chromatograms of a blank plasma and one spiked with 8 Fg/mL ampicillin. The lowest detectable plasma concentration is 0.2 pg/mL, just as predicted theoretically. For comparison we have also used a UV detector set to a wavelength of 230 nm. From the signals obtained and the noise, which is mainly caused by minor interferences from the plasma samples, the lowest detectable concentration can be estimated a t 1-2 pg/mL. When urine samples are analyzed, the sensitivity of the method is not so important, since the ampicillin concentration found in urine after ingestion of a normal dose is quite high. However, as can be seen from Figure 4, the greater selectivity of the bromine generation system over UV detection is a distinct advantage. Less demands are made on the sample preparation procedure and the chromatographic system. Other Penicillins. We have performed some preliminary experiments with other penicillins, penicillin G and amoxycillin. The reaction of bromine with penicillin G is slower than with ampicillin. This might be caused by the higher methanol content (40%)of the mobile phase used to elute penicillin G from a C-18 bonded phase column. A decrease of reaction rates with increasing methanol concentrations has been observed before for other types of reaction (12). Amoxicillin reacts faster than ampicillin. Electrophilic sustitution of the phenol group in amoxicillin by bromine is an explanation for this observation. Through this phenolic
group, detection of amoxicillin by direct oxidative amperometry is also possible (26). An advantage of the present detection method, however, is the absence of electrode passivation problems. For all penicillins examined, it was observed that degradation products (penicilloicacids, the penillic acid of penicillin G) react very fast with bromine. This implies that these degradation products will be detectable in low concentrations. Ranitidine. Kinetic Studies. The reaction rate of ranitidine with bromine was investigated in the same way as for ampicillin. Standard solutions were injected on a 100 x 4.6 mm column packed with LiChrosorb RP-18 ( 5 pm). The mobile phase contained 20% (v/v) methanol, 0.025 M phosphate buffer (pH 2), 0.01 M KBr, 0.01 M TEAC, and 0.5 mM HSA. Ranitidine eluted with a k’value of 3.2. A reaction coil with 12 s hold up time was used. From the areas of the peaks obtained, the amount of bromine consumed was calculated. Even a t low generating currents ( < l o pA) one molecule of ranitidine reacts with two molecules of bromine. This indicates that low detectable limits can be reached. At higher generating currents further oxidation was observed. A surprising result was that peak-height reproducibility for ranitidine was less than that usually observed with other analytes where the relative peak-height variance is within f 2 % . The sensitivity measured over a period of several weeks was 2.1 f 0.3 nA/ng injected (mean f standard deviations, n = 24). Even in one series of standard solution determinations a relative peak-height variance of f 5 % was found (n = 8, 30 ng injected). Chromatography. Figure 5 shows chromatograms obtained with extracts of blank plasma and of a plasma spiked with 22 ng/mL ranitidine. The lowest detectable plasma concentration is about 2 ng/mL, which is an improvement compared to the literature values for UV detection (5-20 ng/mL). However, the extraction procedure was insufficiently reproducible. The use of an internal standard appeared necessary. The recovery of ranitidine measured was 59 i 26% (mean f standard deviation, n = 26), with extreme values of 5 and 104%. No significant relation between plasma concentration and recovery percentage was found. Instead of trying to find a suitable internal standard, we have focused our study on finding a procedure without an extraction step. Therefore, the chromatographic procedure had to be changed. The injection volume was increased from 20 to 100 pL. A 250 x 4.6 nm column, packed with LiChrosorb RP-8 ( 5 pm) was used. The flow rate was set to 2 mL/min. With a mobile phase containing 25% (v/v) methanol, 0.5 M phosphate buffer (pH 2), 0.01 M TMABr, and 1.1mM HSA,
ANALYTICAL CHEMISTRY, VOL. 57, NO. 13, NOVEMBER 1985
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Table 111. Comparison of HPLC Detection Techniques for Ranitidine in Plasma I25nA
detection UV, 320 nm UV, 330 nm UV, 229 nn UV, 320 nm
sample
preparation
extraction (2X) extraction extraction ultra-filtration amperometry, + 1.25 V extraction bromine generation extraction (2X) bromine generation (deproteinization
a
-
0
5
10
15
0
t[min]
5
10 t[min]
15
Flgure 6. Chromatograms obtained with deproteinized plasma: (a) blank plasma; (b) plasma spiked with ranitidine (50 ng/mL).
Table 11. Recovery of Ranitidine from Plasma after Deproteinization ranitidine
added, PLp/mL 0 0.020 0.050 0.100 0.200 0.300
Mean
2583
f
ranitidine found,” Pg/mL
recovery, %
0.006 f 0.006 0.023 f 0.002 0.047 f 0.008 0.082 f 0.012 0.166 f 0.023 0.244 f 0.014
115 94 82 83 81
standard deviation from triplicate injections.
ranitidine eluted with a k’value of 3.7, well separated from other plasma components (see Figure 6). The lowest detectable plasma concentration is about 10 ng/mL. In Table I1 the recoveries after deproteination of the plasma samples are given. The values found are sufficiently close together to eliminate the need for an internal standard. At low concentrations the amount of ranitidine tends to be somewhat overestimated probably by the occasional appearance of small endogeneous peaks close to the elution time of ranitidine (see Figure 6a). In Table I11 a comparison is made between various HPLC determination methods for ranitidine.
CONCLUSIONS In both examples studied the sensitivity of the electrochemical postcolumn reaction system was superior to the UV method now generally employed. The detection limit abtained for ampicillin is comparable to that with other pre- or postcolumn reaction systems. However, no additional sample handling is required, and the extra cost of equipment is low compared to other postcolumn methods. The high sensitivity for penicillin degradation products may be useful for the determination of these compounds in, e.g., fermentation broths or for metabolism studies. For ranitidine determination the main advantage of the present detection method is its selectivity. The elimination of the extraction step seems a major improvement of the sample handling procedure. The applicability of the present method for the detection of other thioethers is mainly determined by the kinetics of the reaction of bromine with the compounds of interest, which are difficult to predict. However, once it is established that a reaction is
limit of detection, ng/mL plasma 10 5 20 20
ref
10-20
21 22 23 27 24
2 10
this work this work
reasonably fast ( k , > lo4),the prospects with regard to sensitivity and selectivity are promising. Preliminary experiments have shown that the system is compatible with precolumn sample concentration and cleanup techniques. The additional conditions for the mobile phase composition (the pH should be below 5, a conducting electrolyte should be added) will not often be a serious problem in finding a suitable separation method. Registry No. Ampicillin, 69-53-4;ranitidine, 66357-35-5;ampicilloic acid, 57457-66-6; penicillin G, 61-33-6; amoxycillin, 26787-78-0; penillic G acid, 13093-87-3;bromine, 7726-95-6.
LITERATURE CITED (1) Rabenstein, D. L.; Saetre, R. Anal. Chem. 1977, 49, 1036. (2) Hanekamp, H. 8.; Bos, P.; Frei, R. W. J . Chromatogr. 1979, 786, 489. (3) Shono, T. I n “The Chemistry of Functional Groups, Suppl. E. Part I ” ; Patai, S. Ed.; Wiley: Chichester, 1980; Chapter 8; pp 327-350. (4) Rao, J. R.; Richter, G. J.; Luft, G.; Von Sturm, F. Biomater., Med. Devices, Artif. Organs 1978, 6 , 127. (5) Reynaud, J. A.; Malfoy, B.; Canesson, P. J . Nectroanal. Chem. 1980, 774, 195. (6) Hui, B. S.;Huber, C. 0. Anal. Chim. Acta 1982, 734 211. (7) Polta, J. A.; Johnson, D. C. J . L l q . Chromatogr. 1983, 6 , 1727 (8) Werkhoven-Goewie, C. E.; Niessen, W. M. A,; Brinkman, U. A. Th.; Frei, R. W. J . Chromatogr. 1981, 203, 165. (9) Barrett, G. C. I n “Comprehensive Organic Chemistry, Vol. 3”; Barton, W., O h , W. D., Eds.; Pergamon Press: Oxford, 1979; pp 33-54. (10) Block, E. I n “The Chemistry of Functional Groups, Suppl. E. Part I”; Patai, S.,Ed.; Wiley: Chichester, 1980; Chapter 13, pp 539-608. (11) King, W. P.; Kissinger, P. T. Clin. Chem. (Winston-Salem, N.C.) 1980, 26, 1484. (12) Kok, W. Th.; Brinkman, U. A. Th; Frei, R. W. Anal. Chim. Acta 1984, 762, 19. (13) Vree, T. B.; Hekster, Y. A.; Baars, A. M.; Van der Kleijn, E. J. J . Chromatogr. 1878, 745, 496. (14) Masada, M.; Kuroda, Y.; Nakagawa, T.; Uno, T. Chem. Pharm. Bull. 1980, 2 8 , 3527. (15) Rogers, M. E.; Adiard, M. W.; Saunders, G.; Holt, G. J . Liq. Chromatogr. 1983, 6, 2019. (16) Miyazaki, K.; Ohtani, K.; Sunada, K.; Arita, T. J . Chromatogr. 1983, 276, 478. (17) Westerlund, D.; Carlqvist, J.; Theodorsen, A. Acta Pt drm. S u m . 1979, 76, 187. (18) Carlqvist, J.; Westerlund, D. J . Chromatogr. 1979, 164, 373. (19) Crombez, E.; Van der Weken, G.; Van den Bossche, W.; De Moerloose, P. J . Chromatogr. 1979, 777, 323. (20) Rogers, M. E.; Adlard, M. W.; Saunders, G.; Holt, G. J . Chromatogr. 1983, 257, 91. (21) Carey, P. F.; Martln, L. E. J . Liq. Chromatogr. 1979, 2 , 1291. (22) Mihaly, G. W.; Drummer, 0. H.; Marshall, A.; Smallwood, R. A.; LOUIS, W. J. J . Pharm. Sci. 1980, 6 9 , 1155. (23) Boutagy, J.; More, D. G.; Munro, I.A.; Shenfield, G. M. J . Liq. Chromatogr. 1984, 7 , 1651. (24) Oosterhuis, B. I n “Progress in HPLC, Vol. 2. Electrochemical Detection in Medicine and Chemistry”; Parvez, H., Parvez, S., Bastart-Malsot, M., Nagatsu, T., Bergstedt, L., Pellerin, J., Eds.; VNU Int. Sci. Press: Utrecht, Holland, in press. (25) Albery, W. J.; Hitchman, M. L. “Ring-Disc Electrodes”; Clarendon Press: Oxford, 1971. (26) Brooks, M. A.; Hackman, M. R.; Mazzo, D. J. J . Chromatogr. 1981, 270, 531. (27) Carey, P. F.; Martin, L. E.; Evans, M. B. Chromatographla 1984, 79, 200.
RECEIVED for review May 28,1985. Accepted June 28,1985.
