Europ. J.clin. Pharmacol. 9, 451-456 (1976) © by Springer-Verlag 1976

Determination of Therapeutic Serum Concentrations of Oral and Parenteral Meperidine by Gas Liquid Chromatography A. P. L. Shih, K. Robinson* and W. Y. W. Au D e p a r t m e n t s of P h a r m a c o l o g y and Toxicology, M e d i c i n e and Dental Research, of R o c h e s t e r School of M e d i c i n e and Dentistry, Rochester, New York 14642,

Received: accepted:

October 8, 1974, July 25, 1975

and in revised

form:

June

18,

University U.S.A.

1975,

Summary. A s e n s i t i v e and specific m e t h o d for e s t i m a t i o n of serum m e p e r i d i n e by gasliquid c h r o m a t o g r a p h y and its a p p l i c a t i o n in a clinical study are described. Patients were given, under d o u b l e - b l i n d conditions, either oral (75 or 150 mg) or intram u s c u l a r (50 or 1OO mg) doses of m e p e r i d i n e for p o s t o p e r a t i v e analgesia. Serum m e p e r i d i n e levels were m e a s u r e d 0.5, I, 2, 3, and 4 hours post-dose. A l t h o u g h wide v a r i a t i o n s of serum m e p e r i d i n e levels were o b s e r v e d among individuals w i t h i n each group for each time period, the i n t r a m u s c u l a r route p r o d u c e d and s u s t a i n e d sign i f i c a n t l y higher serum levels than did the oral route. The m e a n c o n c e n t r a t i o n of m e p e r i d i n e (pg/IOO ml ± S.E.) for 100 mg i.m. was 37.7 ± 3.8 at one hour and 23.4 ± 5.7 at three hours; for 150 mg orally it was 11.9 ± 1.6 at one hour and 11.8 ± 3.2 at three hours. A c o r r e l a t i o n o b s e r v e d b e t w e e n the i n t r a m u s c u l a r dose (in m g / k g body weight) and serum m e p e r i d i n e c o n c e n t r a t i o n attained at one hour indicated that I mg/kg m e p e r i d i n e i.m. will achieve a o n e - h o u r serum level of a p p r o x i m a t e l y 20 ~g/100 ml, an a p p a r e n t m i n i m a l l y effective level for a n a l g e s i a in m o s t patients. Key words: Serum meperidine, tion, p a r e n t e r a l m e p e r i d i n e

gas-liquid chromatography, administration.

A l t h o u g h m e p e r i d i n e has been used clinically as an e f f e c t i v e a n a l g e s i c agent for over 30 years, little i n f o r m a t i o n is a v a i l a b l e c o n c e r n i n g its serum conc e n t r a t i o n in clinical t h e r a p e u t i c use in humans. In p r e v i o u s l y p u b l i s h e d reports, i n v e s t i g a t o r s have m o s t l y used the m e t h y l orange c o l o r i m e t r i c m e t h o d for e s t i m a t i n g plasma m e p e r i d i n e conc e n t r a t i o n in normal v o l u n t e e r s (I) and in patients m e d i c a t e d with the drug before a n e s t h e s i a (2). R e c e n t l y gasliquid c h r o m a t o g r a p h y (GLC) m e t h o d s p r o v i d i n g increased s e n s i t i v i t y and s p e c i f i c i t y have been a p p l i e d in the d e t e r m i n a t i o n of plasma m e p e r i d i n e c o n c e n t r a t i o n s in normal v o l u n t e e r s (3, 4), in sheep (5), and in dogs (6). We d e s c r i b e here the d e v e l o p m e n t of Fellow, China Medical Board Foundation, New York, N.Y.

oral m e p e r i d i n e

administra-

a specific a n a l y t i c a l GLC m e t h o d using a p o l y i m i d e column w i t h a s e n s i t i v i t y of at least 0.025 ~g/ml (2.5 pg/IOO ml) for e s t i m a t i o n of serum m e p e r i d i n e in patients given t h e r a p e u t i c doses for p o s t - o p e r a t i v e pain. We also compare the serum levels attained after o r a l l y a d m i n i s t e r e d m e p e r i d i n e with those of i n t r a m u s c u l a r doses of the drug in these patients and compare these with therapeutic effects to estimate the d e s i r e d m i n i m a l l y e f f e c t i v e serum level and dose of meperidine.

MATERIALS

AND M E T H O D S

Gas-Liquid Chromatography Apparatus. The g a s - l i q u i d c h r o m a t o g r a p h was a P e r k i n Elmer Model 900 e q u i p p e d with a PEP-I GLC data r e d u c t i o n system (4K), a flame i o n i z a t i o n d e t e c t o r using

452 hydrogen and air at flows optimized for maximum sensitivity, and a Speedomax XL68 Series Recorder (Leeds and Northrup). column. A 6 nun O.D. (2.0 mm I.D.) x 180 cm long coiled glass column was packed with 3% POLY 1-110 on 80-100 mesh Gas Chrome Q (Applied Science, State College, Pa.). Operating conditions were as follows: column oven temperature, 190°C; injection port 250°C, detector temperature 27OOC; carrier gas, helium at flow rate of 30 ml/min with inlet pressure of 20 kp/cm2; dual column balancing system, using two 3% POLY I-IO0 columns; electrometer amplifier set at m a x i m u m sensitivity of 0.5 x 10-12 amplification on full scale with output signal attenuation at 4X to 8X as required. Analytical Procedure. Two ml of serum was transferred to a 30 ml glass-stoppered centrifuge tube containing 2.5 ~g of diphenhydramine as an internal standard, made basic with 0.5 ml I N NaOH, and extracted into 10 ml of analytical reagent grade benzene by shaking for 30 min in a mechanical shaker at m e d i u m speed (New Brunswick Model G33). The benzene phase was separated by centrifugation, transferred to another glassstoppered centrifuge tube and re-extracted with 10 ml of O . 1 N HCI by shaking for 30 min. After separation by centrifugation, the acidic aqueous phase was neutralized with I ml of I N NaOH and buffered with 5 ml of phosphate buffer, 0.5 M, pH 10.4. This mixture was then extracted into 10 ml of chloroform by shaking for 30 min. The chloroform fraction obtained after centrifugation was transferred quantitatively to a conical test tube and evaporated to dryness. The residue was reconstituted with 50 ul of chloroform just before analysis. One ~i of the reconstituted c h l o r o f o r m solution was injected for GLC determinations. Quantitation was obtained with the Perkin Elmer PEP-I automated data reduction system using peak area normalization ratios of unknown to internal standard. The PEP-I data reduction system was calibrated for each analytical experiment by extracting, by the method described above, 2 ml of pooled human serum to which had been added 1.25 ~g/ ml meperidine and 1.25 ~g/ml diphenhydramine for determination of response factor, retention time and normalization of peak area. Simultaneous recording of chromatographic peaks were obtained for the purpose of visual confirmation of peaks and retention times; all quantitation was performed by the

on-line PEP-I automated data processing system. The efficiency of the extraction procedure by chloroform, n-heptane, or benzene as initial solvent was evaluated for meperidine using 14C-meperidine (labeled in the N-methyl position), specific activity 4.51 mCi/mmole (Mallinckrodt Chemicals, St. Louis, Mo.) (see Table I). Assay of Serum Meperidine Level in Patients

Blood was collected at 0.5, I, 2, 3 and 4 hours after administration of meperidine to 33 postoperative patients in a controlled study designed to compare the clinical efficacy of orally and intramuscularly administered meperidine. Thirty of the 33 were female; most (27) of them had undergone bilateral tubal ligation. The patients were randomly allocated to one of four treatment groups based on dose and route of administration of meperidine: Group I, 75 mg orally; Group II, 150 mg orally; Group III, 50 mg intramuscularly; Group IV, 100 mg intramuscularly. The study was conducted under double-blind conditions; isotonic saline was given concurrently intramuscularly if oral meperidine was administered, lactose capsule was given orally if meperidine was given intramuscularly. After one hour, if still required for pain relief, the patient was given a standard dose of 75 mg of meperidine intramuscularly at the direction of the investigator (K.R.) who carried out the evaluation of pain relief by interview. The patients were encouraged but not required to allow the study medication three hours in which to act before requesting remedication. The study treatment was thus evaluated as clinically ineffective ("treatment failure") if remedication was necessary within three hours after the initial medication. Written informed consent was obtained from all patients who entered the study. Blood specimens were received for analysis from the clinic coded for each patient; treatment grouping or remedication was not known to the investigators performing the analysis until the study was completed. The serum was separated and kept frozen at ~15°C until the time of analysis. Blood levels of meperidine obtained after remedication were not included in the data analysis. RESULTS Detector-response ratio of meperidine to the internal standard, diphenhydramine, was linear over a meperidine concentration range of 4 to 96 ~g/100 ml

453

Table 1. Recovery of meperidine and 14Cmeperidine added to human serum using different solvents for initial extraction

M: Meperidine B : Diphenhydramine

M/B

1.5

Initial solvent

pH

o

1.0 c 0 O. m

4 8

16

32

I

~

64

14CMeperidine

Meperidine by GLC

36.3

34

Chloroform

i0.4 7.4

58.0

45

n-Heptane

IO. 4

46.8

-

7.4

1.0

Benzene

10.4

71 .O

70.0

Benzene a

10.4

70.0

71 .O

o.s

-"/-"/6~6~/1" 1 J J

Final percent recovery

I 96

Serum Meperidinej ~/100ml

a

Fig. I. Calibration curves for meperidine standards in serum or chloroform. Two ml of serum containing standards was extracted by the method described in the text; the final volume reconstituted was 50 ~i. M/B is the response ratio of meperidine to diphenhydramine (internal standard) and was obtained by peak-area normalization

f o r a 2 - m l s e r u m s a m p l e (Fig. I). Meperidine standards added to pooled serum and extracted by the described procedure compared favorably with s t a n d a r d s d i s s o l v e d in c h l o r o f o r m . Though minimal serum detection level of 0 . 0 2 5 ~ g / m l (2.5 ~ g / I O O ml) w a s achieved, the working standards for precision measurement r a n g e d f r o m 8 to 125 p g / 1 0 0 ml. T h e [ e l a t i v e s t a n d a r d deviation (RSD, S D / x ) , a n e s t i m a t e of p r e c i s i o n of r e p l i c a t e s a m p l e s of meperidine a d d e d to p o o l e d h u m a n s e r u m at .25, 1.25, a n d 2.5 pg/ml, a n d analyzed at varied time throughout the study, r a n g e d f r o m 5.6 to 12% (Table 2). T h e r e t e n t i o n t i m e for m e p e r i d i n e w a s 2.7 m i n u t e s ; f o r d i p h e n h y d r a m i n e , 4.4 minutes. Although a single extraction w i t h c h l o r o f o r m of s e r u m a l k a l i n i z e d to p H 10 r e s u l t e d in r e c o v e r y of 80% of 1 4 C - l a b e l e d m e p e r i d i n e r a d i o a c t i v i t y , t h e a p p e a r a n c e of a n u n i d e n t i f i e d peak n e a r the r e t e n t i o n t i m e f o r m e p e r i d i n e made this single extraction procedure unsatisfactory f o r GLC. F u r t h e r e x t r a c t i o n to r e m o v e o t h e r i m p u r i t i e s a l s o r e s u l t e d in a l o w e r r e c o v e r y w h e n c h l o r o f o r m w a s u s e d as t h e i n i t i a l s o l v e n t (Table I). T h e d o u b l e e x t r a c t i o n p r o c e d u r e u s i n g b e n z e n e as t h e i n i t i a l s o l v e n t y i e l d e d l o w e r r e c o v e r y f o r labeled meperidine but provided for the r e m o v a l of n o n s p e c i f i c interfering peaks near the retention time for meperidine. T h e d u r a t i o n o f s h a k i n g in s e r u m e x t r a c t i o n p r o c e d u r e s w a s f o u n d to a f fect inversely the meperidine/diphen-

5 ~g of diphenhydramine were added to 2 ml of serum for internal standard. 2 zg of meperidine and i x 10-2 ~Ci 14C-meperidine (4.51 ~Ci/mmole) were added to 2 ml of human serum, pH adjusted, and extracted with one of the above solvents. Meperidine was re-extracted into O.i N HCI, the acidic aqueous solution was neutralized and extracted into chloroform after pH adjustment to 10.4 for 14C-activity and GLC estimation for meperidine. Table 2. Precision in measurement of serum meperidine Added to Serum

~g/ml

N

Measured Dg/ml, mean ± S.D.

0.25

20

0.25 ± .03

± 12

1.25

I0

1.33 ± .17

f 6.4

2.75

13

2.75 ± .31

f 5.6

R.S.D., %a

a

Relative standard deviation in percent, S.D./ . Measured meperidine concentration compared to standard curves of meperidine in human serum; extraction and analysis by same methods as for unknown serum.

hydramine response ratio, decreasing w i t h i n c r e a s i n g d u r a t i o n of s h a k i n g . The optimum shaking time for the ext r a c t i o n p r o c e d u r e w a s f o u n d to b e 30 minutes. A c o m p a r i s o n of d i f f e r e n t c o l u m n s , 3% O V - I , 3% 0 V - 1 7 , 3% S E - 3 0 , w i t h t h e 3% P O L Y 1 - 1 1 0 c o l u m n s u s e d in t h i s e x periment indicated that the polyimide column provided for the most sensitive d e t e c t i o n l i m i t of 0 . 5 n g / ~ l s t a n d a r d in c h l o r o f o r m . B o t h t h e OV-I a n d t h e OV-17 columns, at 17OO-210°C, with a c a r r i e r f l o w r a t e of 30 m l / m i n , a t t a i n e d d e t e c t i o n l i m i t s of 50 n g / ~ l ; S E - 3 0 a t t a i n e d a l i m i t o f 25 n g / ~ l u n d e r t h e same conditions.

454 Assay of Therapeutic Concentration of Serum Meperidine The mean serum meperidine level obtained f r o m g r o u p s of p o s t o p e r a t i v e patients g i v e n d i f f e r e n t m e p e r i d i n e d o s e s a n d by d i f f e r e n t r o u t e s of a d m i n i s t r a t i o n are s h o w n in T a b l e 3. T h e n u m b e r of d e t e r m i n a t i o n s f o r e a c h p e r i o d is v a r i a b l e and, a f t e r t h e 2 - h o u r o b s e r v a t i o n p e r i o d , is s m a l l p a r t i c u l a r l y for Group I, II a n d III b e c a u s e of e l i m i n a t i o n of remedication levels from the study data. Mean values were highest in Group IV (100 m g i.m.) at o n e h o u r a f t e r a d m i n i s t r a t i o n a n d l o w e s t i n G r o u p I (75 m g p. o.); t h e h i g h e s t m e a n p e a k l e v e l w a s u s u a l l y a t t a i n e d at o n e o r t w o h o u r s . T h e m e a n b l o o d l e v e l s for m e p e r i d i n e o b t a i n e d at I/2, I a n d 2 h o u r s for G r o U p IV p a t i e n t s w e r e s i g n i f i c a n t l y h i g h e r t h a n f o r b o t h d o s e s of o r a l a d ministration (p