A gas-liquid chromatographic method for the determination of butriptyline in serum

Clin. Biochem. 10, (1) 3-7 (1977)

A Gas-Liquid Chromatographic Method for the

Determination of Butriptyline in Serum HI~LI~NE Department

BRUDERLEIN,

of Biochemistry,

MICHAEL

Ayerst

(Accepted

KRAML

Research July

Laboratories,

Ayerst

Research

A GAS-LIQUID C H R O M A T O G R A P H I C M E T H OD F O R T H E D E T E R M I N A T I O N OF B U T R I P T Y L I N E IN SERUM 1. A gas-liquid chromatographic (GC) method for the analysis of butriptyline in serum has been developed. Quantitation is based on the peak height ratio between butriptyline and promazine used as internal standard. A triple partition provides a "clean" extract. A detection limit of 10 n g / m l is achieved. 2. The usefulness of the method has been demonstrated in bioavailability studies in dogs.

TESTS IN LABORATORY ANIMALS have

indicated that b u t r i p t y l i n e [ F i g . 1, d , l - 5 - ( 3 P - d i m e t h y l a m i n o - 2 ' - m e t h y l p r o p y l ) d i b e n z o [ a , d ] - [ 1,4] c y c l o h e p t a d i e n e ] p o s s e s s e d a p s y c h o p h a r m a c o l o g i c a l p r o f i l e s i m i l a r to t h a t o f o t h e r t r i c y c l i c a n t i d e p r e s s a n t d r u g s ('). I n s u b s e q u e n t s t u d i e s in man, b u t r i p t y l i n e w a s shown to be a clinically e f f e c t i v e a n t i d e p r e s s a n t cs) F o r p h a r m a c o k i n e t i c a n d b i o a v a i l a b i l i t y studies, we n e e d e d a s e n s i t i v e a n a l y t i c a l m e t h o d f o r t h e d e t e r m i n a t i o n of b u t r i p t y l i n e ; b y a n a l o g y to t h e s t r u c t u r a l l y s i m i l a r a n t i d e p r e s s a n t s a m i t r i p t y l i n e and n o r t r i p t y l i n e (~'4':'', we h a v e chosen t h e g a s c h r o m a t o g r a p h i c technique. T h e d e v e l o p m e n t of a gas-chromatographic method for the measurement

Cc: I CH2--CH2-CH2--N(CH3) 2

. .CH3 Bulriptyline

Quebec

23, 1 9 7 6 )

Bruderlein, H., Kraml, M., and Dvornik, D.

CH2--CH--CH2--N(CH3) 2

Montreal,

of b u t r i p t y l i n e a n d i t s a p p l i c a t i o n in b i o a v a i l a b i l i t y s t u d i e s in dogs is r e p o r t e d h e r e w i t h .

CLB1A, 10, (1) 3-7 (1977) Clin. Biochem.

Department of Biochemistry, Laboratories, Montreal, Quebec

a n d D. D V O R N I K

Promazine

Figure 1 - -

Correspondence: Dr. Michael Kraml, D e p a r t m e n t of Biochemistry, A y e r s t Research Laboratories, P.O. Box 6115, Montreal, Quebec, H3C 3J1

MATERIALS AND METHODS

Reagents Column packing : 3.0% SE-30 stationary phase (General Electric, 6C grade) was coated on Gas-chrom Q (Applied Science Laboratories, 80/100 mesh) using the fl]tratdon technique. Eztraction mL~cture: n-heptane (Fisher, spectr~ltalyzed) and isoamyl alcohol are mixed in a proportion of 95:5 (V/V). Butriptyline standards: 5.0 m g of butriptyline hydrochloride was dissolved in 2.0 ml of pyridine; 0.1 of this solution was diluted to 10.0 ml with methanol, to yield a stock solution containing 25 ~g/ml. By adding 50, 100 and 200 ~1 of the stock solution to 5.0 ml of pooled human serum, a calibration curve of 0.25, 0.5 and 1.0 ~g of h u t r i p t y l i n e / m l serum was constructed. A fresh stock solution was p r e p a r e d f o r each analytical run. Internal standard: 5.0 mg of promazine [10-(3-dimethyiaminopropl)-phenothiazine] (Fig. 1) was dissolved i n 2 . 0 ml of pyridine, and 0.2 ml was diluted to 10.0 ml With methanol, yielding a solution containing 50.0 pg/ml. Other reagenfs: Isoamyl alcohol, methanol, pyridine, and chloroform were r e a g e n t grade solvents ( F i s h e r Co.). Dilute 1.0 N sodium hydroxide and 0.1 N sulfuric acid were prepared from Acculute (Anachemia Chemicals Ltd.) or similar vials. All aqueous solutions were i/repared with deionized water. In~rumenhntion A Varian Aerograph gas chromatograph (Model 2100) equipped with flame ionization detectors was used. The instrument was fitted with a 6-foot U-shaped glass column with a 2 m m i.d. and 0.25" o.d. Gas chromatogram scans were obtained on a Varian Aerograph recorder (Model

20). Other equipment included an all-glass r o t a r y flask evaporator (Buchi), a shaker (Eberbach) with adjustable stroke-frequency, holding 40 sample tubes, and a r e f r i g e r ateed International, Model PR-2 centrifuge. Procedure

To 5.0 ml of serum, in a 40 ml glass-stoppered testtube, ~ a r e added 0.1 ml (5.0 ~g) of the internal standard, 5.0 ml of 1,0 N NaOH and 10.0 ml of heptane:isoamyi alcohol. The tube is agited for 20 minutes, care being taken t h a t shaking is not too vigorous (thus avoiding emulsions). I f necessary, phases a r e separated b y centrio fugation a t 1500 r.p.m, for 6 m i n u t e s . A n aliquot (8..0 ml) of- the :upp~er o r g a n i c phase -is ~tr~.ns~r~ed . ~ ~ n g t h e r 40 -ml tube .-(~voiding-tra~sf~r-~f.~a~a~.~:4:--~;he aqueous phase) and, ~a£ter . a d d l t i o n ~ Y ~ - - ~ "~.i

B R U D E R L E I N , KRAML, AND DVORNIK N H~SO4, the tube is again agitated for 10 minutes. The upper organic phase is removed by aspiration and an aliquot (4.0 ml) of the aqueous phase is transferred to a third 4'0 m l tube. To this are added 1.0 ml of 1 N NaOH and 5.0 ml of the extraction solvent and a third partitioning is carried out as des.cribed above. A 4.0 ml aliquot of the clearly separate organic phase is then transferred to a fourth 40 ml tube and the bulk of the solvent is evaporated under reduced pressure in a gently heated flash evaporator. The residue (0.05 - 0.10 ml) is transferred with a disposable pipette to a Reacti-Vial ~ and the tube is rinsed with 0.5 ml acetone. The excess solvents are removed in a stream of .nitrogen to give a residue of approximately 25 ~1, consisting mainly of isoamyl alcohol; 2-3 ~1 aliquots are injected in the gas chromatograph (GC). The GC operating conditions were as follows: Tc, 214°; Ti -~ Td = 275°; helium flow rate, 15 m l / m i n at 66 psi. Before each run, the column was conditioned by injection of a concentrated solution of the standard. Butriptyline is quantitated by comparing the GC response (peak height ratio of butriptyline vs. internal standard) of the unknown with that obtained in a calibration curve of 0, 0.25, 0.5 an 1.0 ~g/ml of butriptyline added to control serum (in triplicate). Using the serum calibration curve, peak height ratios of butriptyline vs. internal standard are calculated and plotted againt weight ratios. The concentration of butriptyline in serum, "spiked" with 1.0-~g/ml of internal standard, is calculated according to £he formula: Butriptyline (~g/ml) =

RESULTS AND DISCUSSION Gas chromatograms T y p i c a l gas c h r o m a t o g r a m s of i) e x t r a c t s of control sera o b t a i n e d a f t e r t h e single or t r i p l e e x t r a c t i o n steps, ii) b u t r i p t y l i n e a n d p r o m a z i n e (used as i n t e r n a l s t a n d a r d ) , a n d iii) of a s e r u m e x t r a c t f r o m a b u t r i p t y l i n e - t r e a t e d dog are depicted i n Fig. 2A-D, respectively. R e t e n t i o n t i m e s of b u t r i p t y l i n e a n d t h e i n t e r n a l s t a n d a r d a r e 8.0 a n d 11.5 m i n (Tc, 214°). The t r i p l e e x t r a c t i o n p r o c e d u r e provided a n obviously " c l e a n e r " c h r o m a t o g r a m t h a n single e x t r a c t i o n (2A versus 2B), not only in the r e g i o n of t h e r e t e n t i o n t i m e s of b u t r i p t y l i n e a n d t h e i n t e r n a l s t a n d a r d , b u t

also in respect to the slowly eluted contaminants which m a y interfere with the chromatograms of subsequent injections. Chromatogram 2 D is typical for serum extracts of dogs or h u m a n subjects receiving butriptyline per os. In addition to the butriptyline peak, a prominent second peak, with retention time of 9.0 min, was u s u a l l y p r e s e n t ; p r e l i m i n a r y s t u d i e s i n d i c a t e t h a t it r e p r e s e n t s the N - d e s m e t h y l metabol~te of butriptyline.

R, × F ×(conc. int. std., total ~g) vol. serum

where ._R,. = "..peak height ratio of the unknown sample, and Fj slope of the line obtained by plotting peak height ratios of butriptyline/intexnal standard versus weight ratios for the calibration curve. Methedologieal studies To assess the degree of purification required for the determination of butriptyline in the serum, samples of dog and human serum pools were extracted once with heptane :isoamyl .alcohol or processed by the triple partition procedure described above. The extracts were reduced to a small volume and injected into: the GC. This was carried out with control serum and with sera with added butriptyHne or internal standard. • To determine the efficiency of the extraction .with heptane:isoamyl alcohol, :pooled h u m a n serum was :'spiked" with 0.05, 0.10, 0~25, 0.50 and 1.00 ~g/ml of butriptyline (in triplicate) and the samples were processed as described above, except that the internal standard was added after the extraction. Recovery was estimated by comparison to a pure standard calibration curve. Reproducibility.: of the method was estimated from the calibration curves obtained with pure and extracted standards. In bo..th :cases, the concentrations of butriptyline were 0.25, 0.50 and 1.0 ~g/ml (in triplicate). Repeated analyses were carried out to estimate the daily and day. to-day variations. The s t a b i l i t y of butriptyline was determined by "spiking" pooled human serum at a concentration of 0.40 ~g/ml. Fi.ve replicate samples were analyzed immediately as well as after 2 and 8 days of storage at room tempera,. ture, refrigerated (0-5 °) or frozen. Bioavoilability studies The relative bioavailability of butriptyline from a slow release formulation, prepared by our Pharmacy Research Laboratory, Rouses Point, N.Y., was compared to "that from regular butriptyline tablets. The study was carried out using 6 beagle dogs, each receiving a single oral dose of 150 mg of either formulation. Blood samples were obtained at 1, 3, 5, 7, 11, 15 and 24 hr after ddsing. A f t e r clotting, the sel'a were separated and all saniples were analyzed for butriptyline as described above;

MtN

MIN

t

|

I~

|

|

12

t

~qlN

I

1U

t

21

|

/

I

t/

I UIN

t/

I

/4

I

Figure 2 ~ Typical GC scans of extracts o[ control dog serum obtained after single (A ) or triple extraction ( B ) ; control dog serum "spiked" with butriptyline and internal standard, promazine (C) and serum from a dog treated with butriptyline (D). Butriptyline and promazine have retention times of 8.0 and 11.5 rain, respectively. Recovery I n practice, b u t r i p t y l i n e a n d t h e i n t e r n a l s t a n d a r d are added to pooled h u m a n s e r u m a n d a c a l i b r a t i o n curve is c o n s t r u c t e d ; u n d e r such conditions, no recovery f a c t o r s a r e r e q u i r e d . However, .we have assessed t h e e f f i c a c y of the e x t r a c t i o n p r o c e d u r e by d e t e r m i n -

GC METHOD :FOR ANALYSIS i n g the recovery of b u t r i p t y l i n e f r o m h u m a n s e r u m w i t h the i n t e r n a l s t a n d a r d added a f t e r t h e e x t r a c t i o n . As shown in T a b l e 1, the a v e r a g e recovery of b u t r i p line by the h e p t a n e :isoamyl alcohol e x t r a c t i o n m i x t u r e was 91.3----- 1.60%. W i t h 5.0 ml s e r u m , t h e recovery was i n d e p e n d e n t of the c o n c e n t r a t i o n of b u t r i p t y l i n e in the 0.05 - - 1.0 p g / m l r a n g e . Good recovery of but r i p t y l i n e was m a i n t a i n e d even a t t h e lowest concent r a t i o n of 0.05 /~g/ml.

"spiked" s e r u m s t a n d a r d s ( F i g . 3B). F o r a n y g i v e n day, the c o r r e l a t i o n c o e f f i c i e n t was R >--- 0.99 ( T a b l e 2 ) ; even on c o m b i n i n g t h e d a t a f r o m t h r e e s e p a r a t e r u n s , R was g r e a t e r t h a n 0.98. T h e s i m i l a r i t y i n t h e a v e r a g e slopes of the r e g r e s s i o n l i n e s of t h e p u r e standard and the extracted standard indicates the s i m i l a r i t y b e t w e e n t h e p a r t i t i o n s of b u t r i p t y l i n e a n d p r o m a z i n e used as i n t e r n a l s t a n d a r d . Despite good day-to-day r e p r o d u c i b i l i t y , a 9 - p o i n t (3 c o n c e n t r a t i o n s , in t r i p l i c a t e ) "spiked" s e r u m c a l i b r a t i o n c u r v e is r u n w i t h each set of analyses.

TABLE1 RECOVERY*

OF BUTRIPTYLINE

FROM

HUMAN

SERUM

TABLE 2 Butriptyline, ~g/ml

Recovery

REGRESSION ANALYSESOF BUTRIPTYLINECALIBRATION CURVES

Added

Found

~

Average -4- S.E.

5.00

4.85 4:94 4.75

97.1 98.8 95.0

97.0 =t= 1.1

2.50

2.10 2.10 2.21

84.1 84.2 90.2

86.2 ± 2.0

1.25

1.20 1.23 1.17

96.0 98.3 92.9

~

95.7 1.6

:0.50

0.44 0.47 0.49

87.0 94.2 97.0

±

92.7 3.0

0.20 0.21 0.23

79.0 84.3 91.8

0.25

Regression line: y. = ax -{- b

91.3 1.6

±

Slope: a ± SE

1 (n -- 9)**

1.81 0.015

- 0.02 ± 0.009

0.998

1946

2 ( n = 9)

1.74 0.019

-

0.03 0.012

0.997

2009

3 (n = 9)

2.00 0.011

0.004 0.006

0.999

4102

4 (n = 9)

1.62 0.015

0.02 0.009

0.998

1441

1.71 0.023

0.092 0.015

0.995

685

1.97 0.019

--0.03 0.012

0.997

1382

0.011 0.007

0.988

1016

0.015 0.007

0.986

903

5 (n = 9)

85.0 ±3.7

Over-all Average Recovery

Exp. No.*

:

(n 6 = 9)

(n = 27)

1.84 0.011

8 (n -- 27)

1.78 0.012

7

*A 5.0 ml aliquot of pooled human serum was used. The promazine internal standard (1.0 ~g/ml) was added after the extraction step and the recovery values apply only for the butriptyline extraction.

Intercept: b Correlation ± SE Coef. R.

-

t test

*Regression-lines for peak height ratios versus concentration ratio, were calculated from triplicate assays of 1.25, 2.5 and 5.0 ~g of butriptyline (with 5.0 t~g of promazine internal standard). Experiments 1 2 and 3 were based on pure standards and 4, 5 and 6 were extracted standards (from 5.0 ml of pooled serum). Experiments 7 and 8 represent averages for pure and extracted standards, respectively. **n = number-of points used in calculating the regression line.

Reproducibility

T h e v a l i d i t y of t h e p e a k / h e i g h t r a t i o m e t h o d f o r q u a n t i t a t i o n depends on the l i n e a r i t y a n d r e p r o d u c i b i l i t y o f t h e c a l i b r a t i o n curves. L i n e a r i t y w a s achieved w i t h both p u r e s t a n d a r d s ( F i g . 3A) a n d w i t h

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.410

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F i g u r e 3 - - C a l i b r a t i o n cwrves o f p u r e ( A ) a n d e x t r a c t e d standard~ (B).

I

I

l

6

B R U D E R L E I N , KRAML, AND DVORNIK

Stability Even after standing at room temperature for 8 d a y s , no d e c o m p o s i t i o n of b u t r i p t y l i n e w a s d e t e c t e d ( T a b l e 3).

• 150 riG. tt0Rt1~. I~L(T5 ÷ IS[] M{;. 5(k~lRl~[} R(L(A$[ fORnt~.~Tl{]tl

TABLE 3 B U T R I P T Y L I N E S T A B I L I T Y IN

HUMAN

SERUM

g

~° Recovery, percent* Day

20 °, Room Temperature

Refrigerator

--15 °, Frozen

100.3 ~- I.I

102.6 ± 1.2

105.4 ± 1.2

102.8 ± 1.9

104.6 ± 3.1

98.7 ± 1.8

0-5°~

J,

o.0oo

~.m~

Booo

~.6OG

It,000

I~000

i$ ~

tl oo0

~4.0~c

HOURS aFTF'R O0~'E

*Recoverms are based on pooled human serum "spiked" with 0.40 pg/ml of butriptyline and analyzed immediately. Each value in the table represents 5 replicates.

F i g u r e ~ - - B u t r i p t y l i n e s e r u m c o n c e n t r a t i o n i n dogs g i v e n p e r os 150 m y o f b u t r i p t y ~ i n e i n the f o r m o f r e g u l a r or slow release tablets.

Application of the method to dog serum covered t h e r a n g e of 0.10, 0.20 a n d 0.40 f t g / m l but r i p t y l i n e . T h e r e g r e s s i o n lines f o r such c a l i b r a t i o n curves were not significantly different from those obtained with higher concentrations. W i t h t h i s technique, b a s e d on a 5.0 ml s e r u m sample, t h e l i m i t of d e t e c t i o n o f t h e m e t h o d is a b o u t 10 n g / m l . T h i s r e p r e s e n t s a p e n d e f l e c t i o n of a b o u t 5 mm, a t t h e m o s t s e n s i t i v e a t t e n u a t i o n s e t t i n g s we employed, and is r e a d i l y v i s i b l e o v e r an o t h e r w i s e s m o o t h b a s e line.

To a s s e s s t h e u s e f u l n e s s o f t h e m e t h o d in c o m p a r a tive b i o a v a i l a b i l i t y s t u d i e s , w e h a v e d e t e r m i n e d t h e p r o f i l e of b u t r i p t y l i n e c o n c e n t r a t i o n s in t h e s e r u m o f dogs g i v e n s i n g l e oral doses o f b u t r i p t y l i n e in t h e f o r m of r e g u l a r o r o f s l o w - r e l e a s e t a b l e t s . A s d e p i c t e d in T a b l e 4 a n d F i g . 4, t h e i n i t i a l b u t r i p t y l i n e p e a k observed after the regular tablets was suppressed and appeared at approximately 7 hr after administration o f t h e slow release f o r m u l a t i o n . B a s e d on t h e AUC~.u h*, t h e r e l a t i v e b i o a v a i l a b i l i t y o f b u t r i p t y l i n e f r o m t h e slow release f o r m u l a t i o n w a s 62% 3.

ACKNOWLEDGEMENT A p p l i c a t i o n to h u m a n s e r a W e t h a n k E. G r e s e l i n f o r h i s g e n e r o u s help w i t h t h e e x p e r i m e n t s in dogs, a n d A g n e s C h a c r a a n d L o u i s Deschamps for assistance.

P r e l i m i n a r y s t u d i e s in n o r m a l m a l e v o l u n t e e r s h a v e d e m o n s t r a t e d t h a t , a t t h e r a p e u t i c doses, t h e s e r u m levels o f b u t r i p t y l i n e w e r e l o w e r t h a n u s u a l l y f o u n d in l a b o r a t o r y a n i m a l s . I n v i e w o f this, w e h a v e s l i g h t l y m o d i f i e d t h e p r o c e d u r e so a s t o i n c r e a s e i t s s e n s i t i v i t y . T h e t a k i n g of a 4 / 5 a l i q u o t of t h e u p p e r p h a s e r e s u l t s , a f t e r t h r e e p a r t i t i o n s , in d i s c a r d i n g 49% o f t h e t o t a l d r u g p r e s e n t . H o w e v e r , b y t r a n s f e r r i n g , a t each p a r t i tion, a s m u c h o f t h e u p p e r p h a s e as possible, up to 90% o f t h i s loss c a n be avoided. W i t h s a m p l e s e x p e c t e d to c o n t a i n low c o n c e n t r a t i o n s o f b u t r i p t y l i n e , t h e i n t e r n a l s t a n d a r d w a s r e d u c e d to 0.4 f t g / m l a n d t h e " s p i k e d " s e r u m - c a l i b r a t i o n c u r v e s

FOOTNOTES 'Before use, all glassware was washed in chromic acid, thoroughly rinsed in distilled w a t e r and dried. =Reacti-vials, purchased from Regis Chemical Co., are 1.0 ml tapered glass vials with teflon-faced screw caps. ~In a pilot study using the same dogs, butriptyline was administered a t higher doses, i.e. at 200 mg as the r e g u l a r tablets and at 375 mg as the slow release formulation. The relative bioavailability of butriptyline from the slow release formulation was 90%.

TABLE 4 S E R U M L E V E L S OF B U T R I P T Y L I N E IN D O G S R E C E I V I N G S I N G L E ORAL DOSES O F R E G U L A R T A B L E T S OR A SLOW R E L E A S E F O R M U L A T I O N .

Butriptyline, re. g/ml Serum Butriptyline Dose per/dog

Study 2 (n ffi 6)

1 hr 0.32* 0.17

150 mg regular Tablets

±

150 mg slow Release Form

0.00 ± 0.00

3 hr

5 hr

0.68 0.51 ± 0.13 ± 0.08 0.06 .4- 0.03

*Average serum levels q- S.E. n = 6). **The regular tablets are taken as 100% "available".

0.22 .4- 0.09

7 hr

i i hr

0.42 ± 0.02

±

0.24 0.03

0.40 .4- 0.07

0.25 ± 0.04

15 hr 0.16 ± 0.02

±

O.16 0.03

AUC

Relative, % Bioavailability

6.47 ± 0.85

100"*

24 hr ±

0.08 0.01

0.06 + 0.02

±

0.44 0.64

62

GC METHOD FOR ANALYSIS The slow release formulation w a s apparently more "available" at higher doses. The A U C o . ~ hr for the 150 m g dose w a s 4.04 ng x hr/ml (Table 4) and for a 375 m g dose one would expect a value of 10.0 ~g x hr/ml. In fact, the AUC0.~hr found w a s 18.21 ~g x hr/ml. The non-linearity in the A U C / d o s e response m a y be explained by the susceptibility of butriptyline to undergo "first pass" metabolism in the liver~s~. Since a certain limited a m o u n t of butriptyline is metabolized during the "first pass" through the liver, w h e n the concentration in the portal circulation is low a larger fraction of the total dose will be degraded and not appear in the systemic circulation. This is the case w h e n a low dose of a sustained release formulation is administered.

7 REFI~ENCES

1. Voith, K. and Herr, F. (1969). Arch. Int. Pharmacodyn. 182, 38. 2. International S y m p o s i u m on Butriptyline. (1971). J. Med. Exp. Clin. 2 (5), 249-343. 3. Braithwaite, R. A. and Widdop, B. (1971). Clinica Chimica A c t a 35, 461. 4. Hucker, H. B. and Stauffer, S. C. (1974). J. Phar~n.

Sc/. 63, 296. 5. Jorgensen, A. (1975). Acta pharmacol, et toxicol. 36, 79. 6. Gibaldi, M., Boyes, R. N., and Feldman, S. (1971). J. Pharm. Sci. 60, 1338; and references cited therein.