Determination of Butaperazine in Biological Fluids by Gas Chromatography Using Nitrogen Specific Detection System J.I. Javaid*, H. Dekirmenjian, U. Liskevych, and J.M. Davis, Illinois State Psychiatric Institute, Department of Research, 1601 West Taylor Street, Chicago, Illinois 60612.

Abstract A simple and sensitive gas chromatographic method for the quantitative determination of butaperazine in biological fluids is described. The use of a nitrogen specific detector reduces the number of interfering peaks, thereby increasing the number of samples that can be analyzed. When butaperazine is extracted from 2 ml of plasma, the coefficient of variation is 7.4% over the concentration range of 5-180 ng/ml. The method was used to measure the levels in plasma and red blood cells in schizophrenic patients treated with butaperazine. It was also extended to measure butaperazine levels in rat red blood cells, plasma, liver, and brain after intraperitoneal injection (15 mg/kg).

Introduction Interindividual variations in plasma levels of drugs among patients receiving similar doses have been reported (1-4). These variations are due to the individual differences in drug uptake, metabolism, and elimination. It has been suggested that there may exist a "therapeutic window" or a range of drug concentration in plasma, below and above which the patient would not clinically respond (5). Thus, it would be useful to monitor drug levels in blood for many types of patients in order to ensure therapeutic success. Butaperazine (BPZ) is a phenothiazine which is used for the treatment of schizophrenia. Although Kapadia, et al. (6) have described a thin layer chromatographic (TLC) method, it is not sensitive enough to measure BPZ in the therapeutic range. At present, the methods commonly used for the determination of BPZ are by fluoromety (7-9). Here, a specific, simple, and sensitive gas liquid chromatographic (GLC) method for the determination of BPZ in human plasma, red blood cells (RBC), rat liver, and brain is reported. In this method, a number of commonly used psychoactive drugs and the two known BPZ metabolites, BPZ sulfoxide and BPZ sulfone, do not interfere with BPZ analysis.

West Germany. Thioridazine (Mellarir, lot #67761) was a gift from Sandoz Pharmaceuticals (East Hanover, New Jersey). All the other reagents used were of analytical grade, expect for heptane and 2-propanol which were of spectral grade (Fisher Scientific Company, Itasca, Illinois). It was found that sometimes, when there were batch variations of some reagents, the stability of the extracted BPZ was affected. However, when sodium diethylthiocarbamate was used (1 mg/ml), the extracted BPZ was stable. This reducing agent was routinely added to the heptane:2-propanol mixture. All glassware was silanized before use to minimize the adsorption of BPZ to glass. A Hewlett-Packard (Rolling Meadows, Illinois) gas chromatograph (5730-A) equipped with a dual nitrogen-phosphorous-flame ionization detector (N/P-FID) was used for analysis. Three percent OV-17, 3 % OV-101, all on 80/100 mesh WHP, were obtained from Supelco (Bellefonte, Pennsylvania). All gases were of highest purity available from Liquid Carbonic (LaGrange, Illinois). The chromatographic conditions were as follows: Air 60ml/min H2 3 ml/min He (Carrier) 30 ml/min Detector temp. 300°C Range 1 Injection port and oven temperatures were varied according to the column used. These conditions are given in Table I.

Table I. Retention Times for BPZ, BPZ-Sulfone BPZ-Sulfoxide Using Different GC Columns.

Temperature (°C)

Column

Injection DetecPort Column tor

Retention Time (min)

BPZ Sulfone

Sulfoxide

300

260

300

0.9

nd

nd

300

250

300

1.5

3.2

3.6

300

285

300

3.5

nd

nd

350

285

300

2.8

5.8

5.5

Butaperazine dimaleate (low #33744) and 14 C-butaperazine dimaleate were provided by A.H. Robins Company (Richmond, Virginia). The two metabolites of BPZ, the sulfoxide and sulfone, also provided by A.H. Robins, were kindly prepared by Drs. Rose and Kobberline of Bayer A.G.,

1% OV-101 (4 ft) 2% OV-101 (3 ft) 3 % OV-17 (2 ft) 3%OV-1 (4 ft)

•Author to whom all correspondence should be addressed.

*nd = not determined.

Materials and Methods

and

Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.

666 • DECEMBER 1979

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Detector voltage was set to give a 10% deflection on the recorder according to the instruction manual provided by the manufacturer. Procedures Blood was drawn in tubes containing disodium ethylenediamine tetraacetate (EDTA) (1 mg/ml blood) from patients who were being treated with BPZ. Vacutainer stoppers were avoided since it has been reported (10,11) that the stopper may alter the distribution of drugs between plasma and RBC. Plasma and RBC were separated by centrifugation and frozen until analysis. Plasma extraction. Two ml of plasma were transferred to a 15 ml test tube and 100 ng of thioridazine was added as an internal standard. Then 0.5 ml of 2 N NaOH was added and the mixture was extracted with 5.0 ml heptane:2-propanol (9:1, v/v). After shaking for 30 min and centrifuging for 5 min at 1,500 rpm, the organic layer was transferred to another 15 ml test tube containing 1 ml of 0.1 N HCL. The mixture was shaken for 10 min and the organic layer was discarded by aspiration. The acid layer was transferred to a 5 ml centrifuge tube and 0.1 ml of 5 N NaOH was added. The sample was mixed and extracted with 0.3 ml of the heptane:propanol after shaking for 5 min and centrifuging as above. Two microliters of the organic layer was then injected into the gas chromatograph. Extraction from red blood cells. Butaperazine from RBC was extracted as described above with the following modifications: to 2.0 ml of RBC, 2 ml of water was added and shaken for 10 minutes. To the same tube 1.0 ml of 2 N NaOH and 5.0 ml of heptane:2-propanol (9:1, v/v) were added, shaken, centrifuged as above, and the organic layer removed. The mixture was extracted with another 5.0 ml aliquot of heptane:2-propanol. The organic layers were then combined and extracted with 2.0 ml of 0.1 N HC1. The acid layer was transferred to a 5 ml centrifuge tube and 0.2 ml of 5 N NaOH was added. The sample was then extracted with 0.3 ml of heptane:propanol mixture and the organic layer was injected into the gas ehromatograph. Extraction from rat liver and brain. Butaperazine, dissolved in 0.1 N HCI and pH adjusted to 5.0, was injected into rats intraperitoneally (15 mg/kg). The animals were sacrificed at different time intervals. Blood was collected in EDTA (1 mg/ml blood) and separated into plasma and RBC for BPZ analysis. Rat brain and liver were removed and homogenized in 0.4 N HCI (1:10, w/v) using a polytron homogenizer (Brinkman, Westbury, New York) at a setting of 6 for 1.5 min. The homogenate was then centrifuged and the acid layer kept frozen at -16°C until analysis. To 2 ml of the acid extract, 1 ml of 4 N NaOH was added and the BPZ was extracted as described in the RBC extraction procedure. Calculations. The standard curve was prepared by adding 100, 150, 200, and 250 ng of BPZ (as free base) and 100 ng of thioridazine (as free base) to drug-free plasma. The samples were extracted and analyzed as described above. The ratio of peak heights for BPZ and thioridazine was used to prepare the calibration curve, and its slope was used to calculate the concentration of BPZ in the samples. Results

80 • x O x

B 60 •

BPZ 40 BPZ

20

A 0

1.0

3.0

RETENTION

1.0

3.0

TIME

1.0

3.0

(minutes)

Figure 1. A typical chromatogram obtained by using a 3 ft column packed with 2% OV-101. For temperature conditions see Table I. The instrument range was set at 1 with an attenuation of 16. (A) Plasma blank carried through the procedure. (B) Drug free plasma to which 100 ng of thioridazine (Th) and 200 ng of butaperazine (BPZ) were added and carried through the procedure. (C) Plasma from a patient on chronic treatment with butaperazine. One hundred ng of thioridazine was added as an internal standard.

OV-101. At 250°C isothermal, butaperazine gave a symmetrical peak with a retention time of 1.5 min. Under the same chromatographic conditions thioridazine also eluted as a symmetrical peak with a retention time of 1.0 min. Figure 1A is the plasma blank carried through the procedure, whereas Figure IB is a plasma sample to which 200 ng of BPZ and 100 ng of thioridazine was added. Figure 1C shows the peak obtained from plasma from a patient who was treated with BPZ. As an internal standard 100 ng of thioridazine was added to that plasma sample prior to analysis. Figure 2 depicts a standard curve calculated from the ratio of peak heights for different concentrations of BPZ to the peak height for 100 ng of thioridazine. This ratio was linear up to 500 ng of BPZ added to the plasma. Similar results were obtained for BPZ analysis in RBC. Accuracy and Reproducibility

Figure 1 shows a typical chromatogram obtained from the analysis of plasma BPZ using a 3 ft column packed with 2%

Recoveries for BPZ and thioridazine were determined by adding varying concentrations of the standard solution to drug-free plasma. These samples were then extracted and analyzed by the described procedure. The recoveries were calculated by comparing the extracted standard with the

JOURNAL OF CHROMATOGRAPHIC SCIENCE • VOL. 17

DECEMBER 1979 • 667

Butaperazine Determination in Human Plasma and RBC

Table II. Accuracy of Butaperazine Determination in Human Plasma.

ng BPZ/ml Plasma Determined ±SEM Added

5 20 40 100 140 180

No. of Determinations

6.9 ±0.4 21.4+1.2 40.3 ±2.2 94.7 ±1.4 138.6+1.8 179.8 ±2.9

4 5 3 4 5 5

CV, %*

12.9 12.7 9.6 2.9 2.8 3.7 X ± SEM7.4±1.8

100

200

BUTAPERAZINE

300 (ng/ml)

°The coefficient of variation (CV) was calculated from results for drug-free plasma containing known concentrations of BPZ and the values determined according to the described procedure.

Figure 2. Standard curve based on the peak height ratio of BPZ/Th. Different concentrations of BPZ and 100 ng of Th were added to drug-free plasma and the samples analyzed as described in the Methods section.

standard curves obtained by injecting the heptane:2-propanol standard solutions of the drugs without extraction. Butaperazine recovery from plasma averaged 69% ± 4% standard error of mean (SEM), whereas the average recovery for thioridazine was 70% ± 3 % SEM. Both BPZ and thioridazine recoveries were linear when different concentrations of the drugs were used. Butaperazine recovery was also determined by adding "C-BPZ (Sp. Act. 2.46 mCi/mmol) to plasma along with 200 ng of BPZ. Final recovery of radioactivity was 69%. Recovery of BPZ from RBC averaged 77% ±2% SEM. The accuracy of the method was determined by adding different concentrations of BPZ to drug-free plasma and analyzing 3 to 5 samples for each concentration according to the described procedure. These results are summarized in Table II. When BPZ was extracted from 2 ml plasma, the within-day coefficient of variation was 7.4% over the concentration range of 5 to 180 ng/ml. Day-to-day precision of the method was determined by analyzing these samples for five consecutive days. The CV was 10.7% ± 2.8% SEM. Specificity

Using 14C labelled BPZ, Bruce et al. (12) have shown that BPZ is metabolized by the hydroxylation of the phenothiazine ring and N-demethylation of the piperazine ring as well as sulfone and sulfoxide formation. In order to ascertain if some of these metabolites interfered in the present method, BPZ sulfone and BPZ sulfoxide were added to plasma or RBC and extracted as described. When injected into the gas chromatograph under the conditions used for BPZ, sulfone and sulfoxide had longer retention times. Figure 3 shows BPZ sulfone and BPZ sulfoxide were separated from BPZ when analyzed on a 4 ft column packed with 3% OV-1. In fact, it was found by gas-liquid chromatographic (GLC) analysis that both sulfoxide and 668 • DECEMBER 1979

Ir-LUR E T E N T I O N

TIME

( m i n u l t i )

Figure 3. Separation of BPZ from its metabolites on a 4 ft column packed with 3% OV-1. (A) Blank; (B) BPZ; (C) BPZ-sulfone, and (D) BPZ-sulfoxide.

sulfone had approximately 2% and 1% BPZ contamination, respectively. Thus, at least sufone and sulfoxide did not interfere with the BPZ determination employing this method. A number of psychoactive drugs were also analyzed under the conditions used for BPZ determination. These results are given in Table III. None of the drugs tested interfered with BPZ analysis. The commonly used tricyclic antidepressants and diazepam required lower temperatures for separation and eluted immediately after the sample injection under the described conditions. To further ensure the specificity of the GLC method, different types of liquid phases were used for chromatography. BPZ eluted as a single symmetrical peak on 1% OV-101, 3% OV-1, and 3% OV-17 columns. These results are summarized in Table I and clearly suggest that the method described here is specific for BPZ determination. Further evidence of specificity was provided when BPZ levels in different rat tissues analyzed after intraperitoneal BPZ administration were compared with those obtained using a spectrofluorometric method. Although BPZ levels as determined by the GLC method and the fluorometric method in rat plasma, RBC, and brain were similar, the BPZ values in JOURNAL OF CHROMATOGRAPHIC SCIENCE • VOL. 17

Table III. Retention Times for Some Psychoactive Drugs Relative to Butaperazine.

Table IV. Plasma and Red Blood Cells Concentrations of Butaperazine in Patients Treated with Different Doses a .

RRTfl

Drug

ng

Acetrophenazine Amitriptyline Butaperazine Desipramine Fluphenazine Haloperidol Imipramine Mesoridazine Nortriptyline Perphenazine Protriptyline Thioridazine Thiothixene

1.63 b 1.00 b 0.60 0.50 b 1.38 b 1.50 b 0.65 2.1

^Retention time relative to BPZ. "Under the gas chromatographic conditions for BPZ analysis these drugs eluted immediately after sample injection. liver obtained by the fluorometric method were higher than the values obtained by GLC (e.g., 6 hr after drug administration fluorometric values were 1200 ± 130 ng/g liver compared to 440 ± 50 ng/g liver by GLC; n = 4, p < 0.01). The higher spectrofluorometric values for BPZ in rat liver could be due to the presence of some hydroxylated metabolites which did not interfere with the GLC method. No attempt was made in the present study to see if this interfering metabolite could be eluted under different chromatographic conditions. Plasma and RBC Levels in Clinical Samples The method described here has been employed for measuring BPZ plasma and RBC levels in patients involved in different studies. In one such study, hospitalized acute schizophrenic patients were treated with 20, 40, or 80 mg BPZ per day given twice in equally divided doses. Butaperazine was given orally at 9:00 AM and 9:00 PM and blood samples were withdrawn at 9:00 AM, prior to the morning medication. The mean BPZ concentrations in plasma and RBC were calculated from 4-5 blood samples during days 4-14 of steady dose drug treatment. Table IV shows these values in 15 patients on different doses of BPZ. There were large individual variations in plasma and RBC levels of BPZ, e.g., plasma BPZ levels of patients receiving 10 mg, twice daily, varied four-fold, ranging from 23 ng/ml to 99 ng/ml (Table IV). Garver, et al. (4) have similarly reported large interindividual variations in plasma BPZ levels. There was a dose-related increase in the mean steady state plasma levels (Table IV). Treatment response in these patients was measured by using a linear regression of the New Haven Schizophrenic Indices (NHSI) scores on day " 0 " (baseline) and day 14 of drug treatment. The relationship between plasma concentration and the degree of improvement suggested a curvilinear relationship. The details of these clinical studies have been reported elsewhere (13). Discussion In 1967, Hammer and Sjoqvist (1) demonstrated that JOURNAL OF CHROMATOGRAPHIC SCIENCE • VOL. 17

Dose per day

Plasma

20 mg

X±SEM 40 mg

X±SEM 80 mg

X±SEM

RBC

Plasma/ RBC Ratio

79 23 45 47 99

26

9 22 24 12

8.3

59 ±12

19±3

3.6±1

99 95 142 79 187

8.3 3.7 3.6 3.0 3.1

120 ± 18

12 26 40 26 61 33 ±7

4.3 ±0.9

113 145 139 250 206

63 88 64 48 61

1.8 1.7 2.2 5.2 3.4

171 ±22

65 ±6

2.8±0.6

3.0 2.6 2.1

1.96

^Patients were treated with butaperazine (10, 20, or 40 mg dose, twice daily) and bloods were drawn for analysis prior to the morning dose. "Each value is an average of 4-5 samples during days 4-14 of steady dose drug treatment.

different patients given the same doses of tricyclic antidepressants had different plasma concentrations. Since then, such interindividual variations for many psychotropic drugs have been well established. It has been suggested that these interindividual variations in plasma steady state levels may affect the clinical outcome of drug therapy (5). Garver, et al (4) have shown that BPZ levels in RBC are related to clinical response in a curvilinear fashion. These observations have necessitated the development of new analytical methods that are sensitive at therapeutic levels of these drugs and are simple enough to use routinely. The method described here for the measurement of BPZ is simple, specific, and reproducible. The use of a nitrogenspecific detection system reduces the number of interfering peaks, thus increasing the number of samples which can be analyzed. Although BPZ could be eluted from different column systems, it was found that 2% OV-101 in a 3 ft column gave the best results. Thioridazine, another phenothiazine, was used as an internal standard because of its similarities to BPZ structure and extraction characteristics from plasma. Furthermore, the peak heights of both BPZ and thioridazine were linear with the amounts injected, and their retention times were close under the chromatographic conditions described here. Although fluphenazine interfered with the internal standard thioridazine, most of the psychoactive drugs tested here did not interfere with BPZ analysis under the described conditions. The precision (CV) of the method was 2.9% for 100 ng/ml BPZ when 2 ml of plasma was used for the analysis. The DECEMBER 1979 • 669

sensitivity of the method was 5 ng/ml BPZ and could be increased by using larger volumes of plasma, although for clinical purposes 2 ml plasma was sufficient when patients were treated with normal doses of butaperazine (20-40 mg per day). Although BPZ values in rat plasma, RBC, and brain, obtained by the fluorometric method were similar to those determined by the GLC method described here, the BPZ values in liver obtained by fluorometric method were higher than the ones obtained by GLC. The higher fluorometric values for BPZ in rat liver were most likely due to the presence of some hydroxylated metabolite which did not interfere with the GLC method. As the two metabolites of BPZ could be separated from BPZ, the method described here could also be extended to study the formation of BPZ-sulfone and BPZ-sulfoxide in vivo. Acknowledgement This research was supported in part by Foundations' Fund for Research in Psychiatry #77-610. References 1. W. Hammer and F. Sjoqvist. Plasma levels of monomethylated tricyclic antidepressants during treatment with imipramine-like compounds. Life Sci. 6: 1895-1903 (1967). 2. B. Alexanderson. Pharmacokinetics of nortriptyline in man after single and multiple oral doses: the predictability of steady-state plasma concentrations from single dose plasma level data. Eur. J. Clin. Pharmacol. 4: 82-91 (1972). 3. A. Forsman, G. Folsch, M. Larsson, and R. Ohman. On the me-

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H Haefelfinger, P Hajlbrahim, S.K., see McFadden, W.H. Hamilton, J.G., see Horn, L.R. Hangac, G., see Hanna, D.A. Hanna, A., J.C. Marshall, and T.L. Isenhour Hanna, D.A., G. Hangac, B.A. Hohne, G.W. Small, R.C. Wieboldt, and T.L. Isenhour Hatch, P.G., see Pierce, H.D., Jr. Hausler, D.W., J.W. Hellgeth, H.M. McNair, and L.T. Taylor Hawkes, E.C., and S.J. Hawkes Hawkes, S.J., see Hawkes, E.C. Hayashi, K., seeTakagi, T. Heller, S.R., see Fisk, C.L. Hellgeth, J.W., see Hausler, D.W. Hill, K.R. and H.L. Crist Hoffmann, J.J., S.J. Torrance, and J.R. Cole Hogge, L.R., see Adams, R.P. Hohne, B.A., see Hanna, D.A. Holder, C.L., W.M. Blakemore, and M.C. Bowman Hopkins, B.J., see Boshoff, P.R. Horn, L.R., L.J. Machlin, and J.G. Hamilton I I lie, A.E., see Kopecni, M.M. Isenhour, T.L., see Hanna. A. Isenhour, T.L., see Hanna, D.A. Itabashi, Y., seeTakagi, T.

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670 • DECEMBER 1979

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tabolism of haloperidol in man. Curr. Ther. Res. 21: 606-17 (1977). 4. D.L. Garver, H. Dekirmenjian, J.M. Davis, R. Casper, and S. Ericksen. Neuroleptic drug levels and therapeutic response: preliminary observations with red blood cell bound butaperazine. Am. J. Physchiatry. 134: 304-07 (1977). 5. J.M. Davis, S. Ericksen, and H. Dekirmenjian. In Psychopharmacology: A Generation of Progress. M.A. Lipton, A. DiMascio, and K.F. Killam, eds., Raven Press, New York, New York, 1978, pp. 905-15. 6. A.J. Kapadia, H.A. Barber, and A.E. Martin. Quantitative determination of butaperazine by T.L.C. J. Pharm. Sci. 59: 1476-79(1970). 7. G.M. Simpson, R. Lament, T.B. Cooper, J.H. Lee, and R.B. Bruce. The relationship between blood levels of different forms of butaperazine and clinical response. J. Clin. Pharmacol. 13:288-99(1973). 8. D.H. Manier, J. Sekerke, J.V. Dingell, and A.K. El-Yousef. A fluorometric method for the measurement of butaperazine in human plasma. Clin. Chim. Ada. 57: 225-30(1974). 9. D.L. Garver, J.M. Davis, H. Dekirmenjian, F.D. Jones, R. Casper, and J. Haraszti. Pharmacokinetics of red blood cell phenothiazine and clinical effects. Arch. Gen. Psychiatry. 33: 862-66(1976). 10. R.C. Veith, V.A. Raisys, and C. Perera. The clinical impact of blood collection methods on tricyclic antidepressants as measured byGC/MS-SIM. Commun. Psychopharmacol. 2:491-94(1978). 11. E. Cochran, J. Carl, I. Hanin, S. Koslow, and E. Robins. Effect of vacutainer stoppers on plasma tricyclic levels: A reevaluation. Commun. Psychopharmacol. 2: 495-503 (1978). 12. R.B. Bruce, L.B. Turnball, J.H. Newmann, J.M. Kinzie, P.H. Morris, and F.M. Pinchbeck. Butaperazine dimaleate metabolism. Xenobiotica. 4: 197-207 (1974). 13. R. Casper, D.L. Garver, H. Dekirmenjian, S. Chang, and J.M. Davis. Plasma and red blood cell phenothiazine levels and their relationship to clinical improvement. Arch. Gen. Psychiatry. In press.

Jaulmes, A., see Colin, H. Javaid, J.I., H. Dekirmenjian, U. Liskevych, and J.M. Davis Jennings, W Jennison, T.A., B.S. Finkle, D.M. Chinn, and D.J. Crouch Johnson, P Jones, G.R., see Coutts, R.T. Julia, S. and J.M. Sans Julian, R.L., see Mattson, D.R. Julian, R.L. see Witt, J.D. Jupille, T Juvet, R.S., Jr., see Wise, S.A.

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K

423

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327

Labastida, C , S. Capella, and A. Manjarrez. Lapointe, M., see Cohen, H. Continued on page 676.

.642

617 285

395 287

91 538

yx.

48 273

137 225

253

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