Chapter III Whole Blood Sample Clean-Up for Chromatographic Analysis

CHAPTER 111

WHOLE BLOOD SAMPLE CLEAN-UP FOR CHROMATOGRAPHIC ANALYSIS U.

CHRISTIANS AND K.-FR. SEWING

Introduction Extraction procedures for blood samples Liquid-liquid extraction Solid-liquid extraction Col umn-switch i ng 3. Blood sample reparation and HPLC analysis o f SandimmunR (cyclosporine! 3.1 Introduction 3.2 Blood sample preparation for SandimmunR (cyclosporine) measurement 3.2.1 Liauid-1 iauid extraction orocedures 3.2.2 Solid-liquid extraction piocedures 3.2.3 Extraction and analysis by col umn-swiiching 3.3 Chromatographic analysis of Sandimmun (cyclosporine) and ts metabolites 4. Trouble shooting in development of blood sample clean-up p ocedures References

1. 2. 2.1 2.2 2.3

INTRODUCTION From all biological fluids, blood is of the greatest analytical interest, since it is the most important transport medium in the human body and blood levels of most therapeutic and diagnostic substances correlate with their function. This chapter is divided into two main parts: in the first part, general strategies for blood sample handling for various drugs are presented: in the second part, as an example measurement of cyclosporine and its metabolites is discussed in detail. Cyclosporine proved to be a good example since blood level measurement is of high clinical impact and the whole spectrum of sample preparation strategies has been applied for cyclosporine determination. 1.

Developing an adequate sample preparation method several factors must be considered: 1. the chemical properties of the constituents

in question.

2. the biological matrix.

The substance of interest must be extracted from its biological matrix prior to chromatographic analysis. Proteins and other macromolecules may interfere with detection and columns may get plugged or rapidly inactivated. A good extraction procedure should be reproducible with little

l o s s o f t h e m a t e r i a l o f a n a l y t i c a l i n t e r e s t . I t should b e r a p i d and a l l o w several samples t o be analyzed i n a s h o r t p e r i o d o f t i m e and i t should be inexpensive. Because o f b e t t e r h a n d l i n g t h e use o f plasma or serum as b i o l o g i c a l m a t r i x i s p r e f e r a b l e over blood. Plasma and serum i s produced by removing t h e c e l l u l a r components o f b l o o d by c e n t r i f u g a t i o n o r n a t u r a l c l o t t i n g . This s t e p must be regarded as a p r e - a n a l y s i s p u r i f i c a t i o n . However, t h e r e a r e some c o n d i t i o n s under which drugs r e q u i r e measurement i n blood r a t h e r than plasma o r serum: 1. The p a r t i t i o n between c o r p u s c u l a r components and plasma depends on

c o n d i t i o n s which cannot be e a s i l y c o n t r o l l e d and/or when t h e drug i s p r e f e r e n t i a l l y bound t o b l o o d c e l l s (e.g. 2. The sample volume i s small (e.g.

cyclosporine).

i n p e d i a t r i c s , experimental animals

o r i n v i t r o systems).

3. Blood samples a r e dehiscent and decomposed so plasma o r serum i s impossible (e.g.

t h a t t h e production o f

i n f o r e n s i c medicine).

4. The drug develops i t s e f f e c t s i n t h e b l o o d c e l l s (e.9.

chloroquine).

5 . Blood l e v e l s r e f l e c t t h e t h e r a p e u t i c and t o x i c e f f e c t s b e t t e r t h a n plasma o r serum l e v e l s (e.g. some drugs a c t i n g on t h e c e n t r a l nervous system) ( r e f . 1 ) . For development of

an e x t r a c t i o n procedure pK,,

p a r t i t i t i o n coef-

f i c i e n t s i n organic solvents and b i n d i n g t o blood components should be a v a i l a b l e . The d i s t r i b u t i o n i n t h e blood components i n f l u e n c e s t h e c h o i c e o f an adequate m a t r i x , Basic drugs o f t e n have a l a r g e volume of tribution

and a r e d e t e c t a b l e

i n blood only

in

low

dis-

concentrations,

e s p e c i a l l y when administered a t low doses (4 mg/kg body weight) ( r e f . 2 ) . Most a c i d i c and amphoteric drugs can be q u a n t i t a t i v e l y determined i n serum, plasma o r u r i n e . They u s u a l l y remain i n t h e i n t r a v a s a l compartment and have a h i g h a f f i n i t y t o plasma p r o t e i n s . Serum i s produced by n a t u r a l c l o t t i n g o f f i b r i n o g e n acquainted w i t h a removal o f hemo- and l i p o p r o t e i n s , which bears t h e r i s k o f l o o s i n g drugs i n t o t h e c l o t . On t h e o t h e r hand plasma, which must be a n t i c o a g u l a t e d , i s r i c h i n l i p i d s and l i p o p r o t e i n s ,

s i n c e these compounds cannot be

removed by c e n t r i f u g a t i o n . F r e s h l y drawn b l o o d w i t h i t s c o r p u s c u l a r components, l i p i d s , l i p o p r o t e i n s and p r o t e i n s r e q u i r e s a p u r i f i c a t i o n s t e p b e f o r e f u r t h e r e x t r a c t i o n , u s u a l l y hemolysis and p r o t e i n p r e c i p i t a t i o n .

84

The f o l l o w i n g steps o f t h e e x t r a c t i o n procedure a r e s i m i l a r t o those used f o r e x t r a c t i o n from plasma o r serum. To remove remaining m a t e r i a l i n t e r f e r i n g w i t h t h e a n a l y s i s u s u a l l y more extended p u r i f i c a t i o n steps a r e requ ired. The e x t e n t of sample p u r i t y r e q u i r e d depends on t h e a n a l y t i c a l system used and how much i m p u r i t i e s t h e d e t e c t i o n system t o l e r a t e s . For blood sample p r e p a r a t i o n v a r i o u s s t r a t e g i e s have been a p p l i e d : liquid-liquid

extraction

and

solid-liquid

extraction

including

column-switch techniques. I n most cases a n t i c o a g u l a n t s a r e added t o b l o o d samples.

Anticoagulants

a c i d - c i t r a t e dextrose

l i k e ethylenediamine t e t r a a c e t a t e (ACD)

c o n t a i n u l t r a v i o l e t - a b s o r b i ng

which can i n t e r f e r e w i t h t h e m a t e r i a l t o be detected

(EDTA)

and

impurities ,

(ref.

3).

The

a n t i c o a g u l a n t can a l s o i n f l u e n c e t h e accuracy o f t h e measurement ( r e f . 4), s i n c e w i t h EDTA a n t i c o a g u l a t e d b l o o d can be p i p e t t e d more r e p r o d u c i b l y than heparin a n t i c o a g u l a t e d b l o o d p a r t i c u l a r l y a f t e r storage f o r several days. The e f f e c t i v e n e s s o f t h e sample p r e p a r a t i o n procedure depends on t h e completeness o f p r o t e i n removal. Thus a s t e p o f hemolysis and p r o t e i n p r e c i p i t a t i o n i s e s s e n t i a l f o r a l l e x t r a c t i o n procedures from blood, plasma and serum. However, although i t i s almost mandatory t o remove a l l i n e r t proteins,

t h e components o f a n a l y t i c a l

i n t e r e s t should be recovered.

Hemolysis w i t h o u t p r o t e i n p r e c i p i t a t i o n i s achieved by

1. freezing (<-2O"C) 2. ultra-sound

and subsequent thawing o f t h e sample,

3. osmotic shock.

Other techniques 1 i k e mechanical membrane d i s r u p t u r e and enzymes a r e seldom i n blood a n a l y s i s . The f o l l o w i n g d e p r o t e i n a t i o n techniques a r e used ( r e f . 5 ) : 1. Change i n pH by adding a s t r o n g a c i d t o t h e sample (e.g.

c h l o r o a c e t i c a c i d , p e r c h l o r i c a c i d , h y d r o c h l o r i c acid)

.

tri-

2. Change i n t h e i o n i c s t r e n g t h by a d d i t i o n o f s a l t s (e.g. ammonium sulfate). 3. Change i n temperature by h e a t i n g t h e sample and d e n a t u r a t i n g t h e proteins. 4. Change i n t h e d i e l e c t r i c constant by a d d i t i o n o f o r g a n i c s o l v e n t s (e.9.

a c e t o n i t r i l e , methanol, e t h a n o l ) .

5. F i l t r a t i o n and u l t r a - f i l t r a t i o n .

a5 During o r a f t e r p r o t e i n p r e c i p i t a t i o n t h e pH r e q u i r e d f o r t h e ext r a c t i o n procedure has t o be adjusted. 2.

EXTRACTION PROCEDURES FOR BLOOD SAMPLES

2.1

LIQUID-LIQUID EXTRACTION

Blood sample p r e p a r a t i o n procedures u s i n g l i q u i d - l i q u i d e x t r a c t i o n can be d i v i d e d i n t o f o u r main steps: 1. hemolysis and p r o t e i n p r e c i p i t a t i o n ,

2. e x t r a c t i o n o f t h e components o f i n t e r e s t ,

3. p u r i f i c a t i o n and removal o f i n t e r f e r i n g m a t e r i a l s , 4. volume r e d u c t i o n and r e c o n s t i t u t i o n f o r chromatographic a n a l y s i s . Generally d e p r o t e i n a t i o n and hemolysis a r e associated w i t h pH adjustment f o r t h e f o l l o w i n g e x t r a c t i o n s t e p i n t o an o r g a n i c s o l v e n t . Thus t h e method chosen f o r d e p r o t e i n a t i o n depends on t h e pKa of

the

m a t e r i a l t o be analysed. A c i d i c drugs a r e e x t r a c t a b l e a t pH < 5.5

and

b a s i c drugs a t a pH > 5.5. I n blood one f r a c t i o n o f drugs i s bound t o plasma p r o t e i n s and t h e o t h e r blood components and t h e o t h e r f r a c t i o n i s free. By d e p r o t e i n a t i o n and e x t r a c t i o n t h e p r o t e i n bonds must be broken o r t h e recovery may be decreased. A decrease i n recovery can a l s o occur if t h e compounds o f i n t e r e s t a r e c o - p r e c i p i t a t e d o r p h y s i c a l l y entrapped i n the protein precipitate.

Basic drugs can be e x t r a c t e d from b l o o d

w i t h o u t p r i o r procedures by t h e use of a p p r o p r i a t e b u f f e r s o l u t i o n s w i t h a pH ranging from 6 t o 14. U s u a l l y t h e pH t o be chosen i s 3 u n i t s above t h e pKa because then more than 99% of t h e b a s i c drug i s i n i t s u n i o n i z e d form and can be e x t r a c t e d i n t o an o r g a n i c solvent.

Due t o t h e i r i o n i c

s t r e n g t h s these b u f f e r s o l u t i o n s cause p r o t e i n d e n a t u r a t i o n w i t h minimal

loss o f t h e drug (refs. a d j u s t i n g t h e pH < 5.5.

2,6,7). A c i d i c drugs can be e x t r a c t e d a f t e r The low pH causes p r o t e i n p r e c i p i t a t i o n w i t h t h e

r i s k o f c o - p r e c i p i t a t i n g t h e compounds of i n t e r e s t . For l i q u i d - l i q u i d p u r i f i c a t i o n o f blood samiles f o u r s t r a t e g i e s a r e used: 1. The drug i s converted i n t o i t s i o n i z e d form by changing t h e pH and can be e x t r a c t e d i n t o an aqueous phase. The o r g a n i c l a y e r i s removed and discarded. I n a second s t e p t h e l i p o p h i l i c , u n i o n i z e d form o f t h e drug i s r e - c o n s t i t u t e d by changing t h e pH i n t o t h e o p p o s i t e d i r e c t i o n and t h e drug can be back-extracted i n t o an organic s o l v e n t ( F i g . 2, s i d e columns)

.

86

2. The drug is dissolved in an aqueous/organic solvent, e.g. water/ acetonitrile, and the interfering compounds are removed by washing the sample with a lipophilic solvent, that is not miscible with the aqueous layer e.g. hexane. Compounds of interest dissolved in a lipophilic solvent can also be purified by washing with an aqueous solution (Fig. 1, middle column).

Liquid-liquid extraction can be combined with solid-liquid purification steps: 1. The extracted sample is spread on a TLC plate. After development the circle of silica adsorbing the compounds of interest is scraped off the plate and the silica gel is extracted (ref. 8). 2. Interfering materials are removed by adsorption on a solid phase or after liquid-liquid extraction the compounds of interest are adsorbed on a solid phase and thus separated from interfering materials.

Fig. 1 i s a schematic flow-diagram of steps used for liquid-liquid sample clean-up of basic and acidic drugs. The diagram demonstrates the way of blood sample preparation for GC and HPLC analysis. For reversed phase chromatography the aqueous back-extracts can be directly injected into the HPLC system The side columns of the diagram show purification steps by basic or acidic back-extraction, the middle column the removal of interfering materials with an organic solvent, inmiscible with the aqueous layer containing the compounds of interest, in which these materials are insoluble. Table 1 shows an overview of blood sample extraction strategies with subsequent chromatographic analysis. The extraction procedures 1 isted are used for sample preparation prior to chromatographic analysis (HPLC, GC, TLC). Usually the authors describe the extraction from several different kinds of biological matrices but only procedures and values for blood sample preparation and analysis are listed here. The procedures are divided into the four main steps as discussed above. In addition de Silva (refs. 9 and 10) describes schematically the extraction of various 1,4-benzodiazepines and Foerster et al. (ref. 11) the extraction of multiple acidic and neutral drugs from blood. Basic back-extraction describes the following procedure: the sample is acidified and the drugs are extracted into an aqueous phase. Then the pH is raised and the drugs are re-extracted into an organic solvent. If the solvents used for extraction and back-extraction are identical the solvent used for back-extraction is not mentioned.

a7

rn

blood sample c o n t a i n i n g a c i d i c druas

blood sample c o n t a i n i n g

protei n precipitation (pH < 5.5)

protein denaturation

I I

extraction into’ organic solvent

e x t r a c t i o n i n t o organic s o l v e n t shake,

I centrifuge

I

shake, c e n t r i f u g e

I I

I

9

d i s c a r d aqueous 1ayer

d i s c a r d a ueous l a y e r

pH r a i s e d > 7 + organic s o l v e n t

+ orga’nic s o l v e n t

decrease pH < 5.5 + organic s o l v e n t

shake,

shake, c e n t r i f u g e

shake, c e t r i f u g e

I centrifuge I

I I

I I

d i s c a r d organic l a y e r

discard organic layer

discard organic layer

decrease pH < 5.5 + organic s o l v e n t

HPLC,‘ GLC

increase PH > 7 + organic s o l v e n t

I

I

I I

I

shake, 2ent r i f uge

shake, c e r h r i f u g e

d i s c a r d aqueos l a y e r

d i s c a r d a ueous l a y e r

I

I I

evaporate o r g a n i c l a y e r -evaporate reconstitution

9

organic layer

I

HPLC. GLC Fig. 1

L i q u i d - l i q u i d clean-up procedures f o r blood sample a n a l y s i s .

rABLE I

m m

Liquid-liquid extraction of blood samples

Substance ( s )

pKa Ref. Deproteinization/ pH adjustment

Acebutolol

9.4 12

l-a acetyl-methadol Acetazol amide Ant i pyrine

Extraction

Purification/ Derivatization

%

rec. Analysis/ Detection

det. .g/l

distilled water, 2 N NaOH

ethylacetate

acidic backextraction

n.r. HPLC / UV (240 nm)

10

pH 9.2

n-butylchloride

basic back-extraction with CHCl,

>90 GC / MS

5

7.2 14 9.0

acetate buffer pH 5

CH C1 /diethylether methylation with /2zprGpanol (6/4/2) trimethylphenylammonium hydroxide

80- Gd3NiECD 90

25

15

filtration, ethanol

CH2C12/n-pentane (50/50) n-butanol/cyclohexane (70/30)

9095 HPLC / UV 9.9 (254nm)

6

13

N ammonium-

Atenolol

9.6 16

Bacmecillinam

6.8 17

CH3C1 /hexane (1/9)

Barbiturates

4.0 18

CH2C1

acidic back-extrac- 83- GC/FID tion, methylation 113

Barbiturates

4.0 19

acetone/ether (50/50)

derivatization with methyl iodide and KJO,

Benzodi azepine derivative

20

0.1

hydroxide

phosphate buffer PH 9

diethyl ether/ CH3C1 (70/30)

basicbackextraction

55

GC/63NiECD

96- HPLC/UV 104 (230 nm)

70

GC/FID

925 HPLC/UV 5.4 (230 nm)

10 0.6 100 16 ng/l

50

TABLE I

(continued) 21

Benzodiazepine derivatives Butaperazine

1

Carbamazepine

22

Carprofen

23

Chloroprocaine

8.7 24

distilled water, ammonium hydroxide

diethyl ether

70- HPLC / UV 100 (240 nm)

n.r.

distilled water, sodium carbonate

n-pentane/ isopropanol (97/3)

82- HPLC/UV 95

<40 n.r.

CH2C1

hexane wash, methyl ati on

94+ 12

acetate buffer pH 5

diethyl ether

TLC, deri vati zati on

42% HPLC/UV 83 (254 nm)

1.8% barium hydroxide

CH3C1

GC/FID

270

94105

GC/FID

10 25

Chl oroquine

8.4 25 10.8

buffer pH 10

diethyl ether

basic back-extraction n.r. from heptane

GC/FID

Chl oroquine

8.4 26 10.8

distilled water NaOH (pH >11)

hexane/ 1-pentanol (90/10)

basic back-extraction 85in CHC13 105

GC/NSD

8.4 27 10.8

distilled water, dipotassium hydrogen phosphate pH 9.5

CH2C12

acidic extraction into the aqueous phase

7397

HPLC/UV (254 nm)

3-4

8.4 28

deionized water, 0.001 N HC1

hexane

basic backextraction

95103

GC/NSD

5-15

6.8 29

freeze and thaw, 1 N NaOH pH 9.0

1-octanol

basic back-extraction 98in ethanol 106

Chl oroquine

Chloroquine Cimetidine

10.8

10

nMol /1

HPLC/UV (228 nm)

50

cn

W

TABLE

I

(continued)

L o

0

Clonazepam

1.5 10.5

30

b o r a t e b u f f e r pH 10

isoamyl a l c o h o l / hexane (10/90)

hydrolysis

3550

GC/ECD

5

Debri soqu ine

11.9

31

d i s t i l l e d water

diethyl ether

b a s i c back-extraction n.r. i n cyclohexane

GC/FID o r NSD

3

Diazepam

3.3

32

1 M phosphate b u f f e r pH 7.0

diethyl ether

b a s i c back-extraction 91116

HPLC/UV (240 nm)

20 30

Diazepam

3.3

33

phosphate b u f f e r pH 7.0

n-heptane

b a s i c back-extraction 9095

GC/63NiECD

D i py r idamol e

6.4

34

sodium hydroxide

diethyl ether

93.9 HPLC/FD(285/ 1 430 nm)

n-heptane

98- GC/TCD 100

pMo 1

Enf 1urane

35

50

4.1

/1

Flestolol

4.0 36

acetonitrile

acetoni t r i 1e/ CH2C12 ( U 5 )

acidic extraction i n t o aqueous phase

38

HPLC/UV (229 nm)

10

Tetrahydrocannabinol

10.6

37

2 N HC1 (pH 4)

hexane/ iso-amyl a1coho1 (98/2)

a c i d i c back-extract i o n i n t o hexane

n.r.

TLC/FD

0.4

116-9-tetrahydrocannabinol

10.6 10.4

38

CHC13

f i l t r a t i o n , TLC

98- GC/MS 100

diethyl ether

e x t r a c t i o n o f the organic l a y e r i n t o a c e t o n i t r i l e and phosphoric a c i d

85

Hydroxychloroquine

39

d i s t i l l e d water, ammonia (pH 13)

0.5

1 HPLC/FD (337/370 nm)

TABLE I

(continued)

Imipramine

9.5 40

ammonium hydroxide

butanol/hexane (20/80)

92.5 HPLC/FD 25 (240/370nm)

Ketamine

7.5 41

ammonium hydroxide pH 10.1

CHC13: isopropanol (75:25) isopropyl acetate

n.r. GC/FI

100

86% GC/MS 9

3

Mef 1 oquine

42

freeze and thaw

Mef 1 oquine

43

phosphate buffer pH 7.4

ethyl acetate

Mefloquine

44

0.2 N H2S04

diethyl ether

wash acidified sample with ether, deri vati zation

1005 HPLC/UV 9.9

935 GC/63Ni ECD 9.7 and FID

50

1

Methadone

8.3

45

4 M Na2C03

1-chlorobutane

basic back-extraction 935 GC/FID into CHC13 2

5

Morphine

8.0

46

phosphate buffer pH 8.7 - 9.0

ethyl acetate

aluminium oxide, deri vatization

1

47

acetate buffer pH 5 , B-glucuronidase

ethylacetatelisopropanol (90/10)

basic back-extraction 81

7.9 48

1 M carbonate buffer pH 10

diethyl ether

distilled water, 5M HC1 glacial acetic acid

Morphine-3glucuronide Naloxone Pentacaine Phenobarbital , Phenytoin, Primidone

9.9

49 7.4 50 8.3

83

GUb5Ni ECO HPLC/ECD

0.5

acidic extraction in aqueous phase

78+ HPLC/UV 3.2 (214 nm)

1

1,2 dichloroethane

heptane, Nap C03 fi 1 trati on

75- G U M S 92

5

CHC13

basic back-

90- HPLC/UV 110 (254 nm)

extraction

100

200

300

ID

c

TABLE I

W

(continued)

N

Phentolamine

7.7

51

1 M amnonium hydroxide

diethyl ether

acidic extraction in aqueous phase

83

Promethazine

9.1

52

borax buffer pH 10

n-heptane/isopentanol (99/1)

basic backextraction

97- GC/NSD 99

5

Propranolol

9.5

53

5 N NaOH

isoamyl-alcohol: nheptane (1.5: 98.5)

n.r. HPLC/FD

5

54

4.8 M KC1 pH 6.1

benzene

98 5 GC/ECD 8.9

2

glacial acetic acid

CHC13

90- GC/FID 110

1000

phosphate buffer pH 5.5

CH2C1

n.r. HPLC/UV (290 nm)

200

67- HPLC/UV 90 (225 nm)

0.03 ppm

Pyramidobenzazepine Theophylline Thiopental

c1 55 8.1

56

Tocainide

7.8 57

1 N NaOH, destilled water

CH3Cl

Warfarin

5.0 58

5 N HC1

CHCl

deri varization

fi 1 tration , wash with aqueous sodium pyrophosphate

HPLC/UV (280 nm)

HPLC/UV (270 nm)

ECD: electron capture detector, FD: fluorescence detector, FID: flame ionization detector, GC: gas chromatography HPLC: high performance 1 iquid chromatography, MS: mass spectrometry, NSD: nitrogen selective detector, TCD: thermal conductivity detector, TLC: thin layer chromatography, UV: ultraviolet absorbance detector, n. r. : not reported.

15

Another f orm o f l i q u i d - l i q u i d e x t r a c t i o n i s t h e use o f s i l i c a m a t e r i a l l i k e E x t r e l u t R ( r e f s . 59-62). Though t h e e x t r a c t i o n columns c o n t a i n s o l i d phase m a t e r i a l , t h e b a s i c p r i n c i p l e i s a l i q u i d - l i q u i d e x t r a c t i o n . E x t r a c t i o n w i t h diatomaceous e a r t h obeys t h e same b a s i c mechanism ( r e f s . 63-65).

Silica

gels

are

porous

carrier

materials.

Water

molecules

d i s t r i b u t e on t h e s u r f a c e of t h e s i l i c a g e l and become t h e s t a t i o n a r y phase. Compounds a r e d i s s o l v e d i n t h e w a t e r phase and a r e e l u t e d f rom t h e columns by o r g a n i c s o l v e n t s , u n m i s c i b l e w i t h wat er. Such columns can be used a t a pH range f r o m 1-13. A f t e r p r o t e i n p r e c i p i t a t i o n by a c i d o r b u f f e r t h e aqueous b l o o d sample i s p u l l e d by vacuum t h r o u g h t h e column ( r e f s . 61,62). S i l i c a g e l can a l s o be used f o r sample p u r i f i c a t i o n ( r e f s . 46,59) by a bs or b i n g i n t e r f e r i n g m a t e r i a l s from b l o o d w i t h o u t absorbing t h e components t o be e l u t e d . 2.2

SOL ID-L I Q U ID EXTRACTION Several methods f o r t h e e x t r a c t i o n o f compounds f rom b l o o d have been

r e p o r t e d u s i n g s o l i d s o r b e n t s as an a l t e r n a t i v e t o l i q u i d - l i q u i d extraction.

F o r t h e e x t r a c t i o n of b l o o d samples t h e use o f s o l i d phase

m a t e r i a l has t h e f o l l o w i n g advantages ( r e f s . 66,67): 1. The f o r m a t i o n o f emulsions d i s t u r b i n g e x t r a c t i o n i s avoided. 2. L i t t l e volume o f s o l v e n t s a r e necessary. 3. A c i d i c drugs can be e x t r a c t e d w i t h h i g h r e covery. 4. F a t t y a c i d s , t h e i r e s t e r s and c h o l e s t e r o l a r e n o t c o - e x t r a c t e d .

The s o l i d - l i q u i d e x t r a c t i o n procedures o f b l o o d samples can be d i v i d e d i n t o 4 main s t ep s : 1. hemolysis, d e p r o t e i n a t i o n and pH-adjustment,

2. a d s o r p t i o n o f t h e compounds of i n t e r e s t on t h e s o l i d phase m a t e r i a l , 3. p u r i f i c a t i o n b y washing t h e adsorbent w i t h l i p o p h i l i c o r h y d r o p h i l i c solvents, 4. e l u t i o n o f t h e drugs f r o m t h e adsorbent, 5. volume r e d u c t i o n and i f necessary d e r i v a t i z a t i o n . L i q u i d - l i q u i d e x t r a c t i o n of a c i d i c drugs i s sometimes complicat ed by the co-extraction

o f 1 i p i d s and 1 i p o p r o t e i n s .

Co-extraction

of

these

compounds i s l e s s i n s o l i d - l i q u i d e x t r a c t i o n and t h u s f u r t h e r clean-up st e ps 1 ike b a c k - e x t r a c t i o n s

o f t e n r e d u c i n g recovery a r e u s u a l l y n o t

r e q u i r e d . For s o l i d - l i q u i d e x t r a c t i o n t h e f o l l o w i n g s o l i d sorbent s a r e used :

94 1. Bonded phase s i l i c a gels, a l s o a v a i l a b l e as pre-packed disposable columns ( r e f s . 68-70), 2. anion and c a t i o n exchange r e s i n s ( r e f . 71), 3. n o n - i o n i c r e s i n s l i k e a c t i v a t e d charcoal ( r e f . 72) and A m b e r l i t e XAD-2 ( r e f s . 73-75)

.

Blood samples must be prepared f o r e x t r a c t i o n on bonded phase s i l i c a gel columns by hemolysis and p r o t e i n p r e c i p i t a t i o n

b e f o r e sucked by

vacuum through t h e e x t r a c t i o n columns. The columns a r e p r e v i o u s l y primed w i t h t h e same s o l v e n t , as used f o r e l u t i o n o f t h e drugs from t h e columns, i n o r d e r t o remove i n t e r f e r i n g substances. With a p o l a r s o l v e n t ( u s u a l l y water) t h e c o n d i t i o n s used f o r e x t r a c t i o n a r e e s t a b l i s h e d by l o a d i n g t h e columns with p o l a r groups. The drugs a r e r e t a i n e d by t h e column and can f u r t h e r be p u r i f i e d by washing t h e adsorbed m a t e r i a l s w i t h l i p o p h i l i c o r h y d r o p h i l i c s o l v e n t s i n which t h e y have a small p a r t i t i o n c o e f f i c i e n t . The columns are a l s o s u i t a b l e f o r an e x t r a c t i o n by i o n - p a i r chromatography ( r e f . 68) and can be cleaned and reused. The l i f e span of e x t r a c t i o n columns used f o r e x t r a c t s from blood i s s h o r t e r than f o r those from serum o r plasma,

s i n c e l a r g e amounts o f blood components

like

l i p o i d s and l i p o p r o t e i n s a r e c o - e x t r a c t e d and can p l u g t h e columns. The columns should n o t be c o n f r o n t e d w i t h s o l v e n t s w i t h a pH > 9. The ext r a c t i o n procedure w i t h disposable s o l i d phase e x t r a c t i o n columns can be automated u s i n g e x t r a c t i o n systems such as an advanced automated sample processing u n i t (AASP,

Varian, Walnut Creek, CA, USA). The p r e - e x t r a c t e d

blood samples a r e a u t o m a t i c a l l y loaded on t h e e x t r a c t i o n columns, p u r i f i e d and i n j e c t e d i n t o t h e HPLC-system ( r e f s . 76,77). I o n i c and non-ionic r e s i n s can be added t o t h e b l o o d sample i n ext r a c t i o n columns ( r e f s . 78-80), bags ( r e f .

i n capsules ( r e f . 69),

78) o r as r e s i n s l u r r y ( r e f s . 74-75).

i n nylon f a b r i c

Anion exchange r e s i n s

are s u i t a b l e f o r t h e e x t r a c t i o n of a c i d i c drugs such as b a r b i t u r a t e s , sal i c y l a t e s and phenylbutazone, c a t i o n exchange r e s i n s f o r t h e e x t r a c t i o n of basic drugs such as q u i n i d i n e , chlorpromazine, s t r y c h n i n e , and morphine. Charcoal i s i n e f f e c t i v e i n b i n d i n g most b a s i c drugs except s t r y c h n i n e and proved v a l u a b l e i n b i n d i n g n o n - i o n i c o r g a n i c compounds l i k e gluthetimide,

meprobamate and carbromal

(ref.

71).

I o n exchange

r e s i n s are a l s o used t o remove i o n i c i m p u r i t i e s from b l o o d samples ( r e f . 81)

.

The a n i o n i c r e s i n Amber1 i t e XAD-2, i n t r o d u c e d i n t o pharmacological and t o x i c o l o g i c a l a n a l y s i s by F u j i m o t o e t a l . ( r e f . 82) i s a nonpolar

styrene-divinylbenzene copolymer w i t h a p a r t i c l e s i z e o f 50-100 p ( r e f . 66).

It a l l o w s t h e e x t r a c t i o n of

deproteination

( r e f . 80).

The

drugs from b l o o d w i t h o u t preceeding

resin

s l u r r y i s prepared by washing t h e

95 r e s i n subsequently w i t h water, methanol and acetone. The r e s i n i s s t o r e d i n water o r a b u f f e r s o l u t i o n ( r e f s . 66,73,75).

A f t e r adsorption o f the

compounds o f i n t e r e s t t h e XAD-2 p a r t i c l e s a r e f i l t e r e d and e x t r a c t e d w i t h an organic s o l v e n t . S c h l i c h t e t a l . ( r e f . 66) and I b r a h i m e t a l . ( r e f . 79) described t h e e x t r a c t i o n o f several drugs from b l o o d samples u s i n g XAD-2 r e s i n s , Ford e t a l . t h e e x t r a c t i o n o f a c i d i c drugs from blood u s i n g CI8 bonded s i l i c a columns ( r e f . 67) and Missen e t a l . ( r e f . 75) compared t h e e x t r a c t i o n of benzodiazepines w i t h various r e s i n s . The e x t r a c t i o n o f drugs from b l o o d using s o l i d phase m a t e r i a l s i s acquainted w i t h some disadvantages t h a t must be taken i n t o account. 1. The e x t r a c t i o n may g i v e v a r i a b l e r e c o v e r i e s

o f the e l u t i n g solvent

and

due t o t h e pH and n a t u r e

t h e sorbent.

2 . The r e s i n s and column m a t e r i a l s loose t h e i r a d s o r p t i o n e f f i c i e n c y t h e

more o f t e n t h e y are reused. 3. The f r i t s and t h e column m a t e r i a l can b e plugged by n o t s u f f i c i e n t l y

d e p r o t e i n i z e d samples o r i f t h e columns a r e reused t o o o f t e n . Since i t i s o f t e n n o t p o s s i b l e t o perform ' d i g i t a l chromatography' on t h e e x t r a c t i o n columns, an i n t e r n a l standard may h e l p t o c o r r e c t recovery o f a compound and t o make e x t r a c t i o n more r e l i a b l e and r e p r o d u c i b l e . A good i n t e r n a l standard should 1. s t r u c t u r a l l y be as s i m i l a r t o t h e compound o f i n t e r e s t as p o s s i b l e , 2. have t h e same d i s t r i b u t i o n c o e f f i c i e n t s i n organic s o l v e n t s ,

3. have t h e same b i n d i n g c h a r a c t e r i s t i c s t o t h e blood compounds as t h e compound o f i n t e r e s t , 4. have a r e t e n t i o n time i n chromatographic a n a l y s i s c l o s e t o t h e compound o f i n t e r e s t , 5. be c l e a r l y separated from t h e compound o f i n t e r e s t d u r i n g a n a l y s i s , 6. have t h e same p r o p e r t i e s concerning t h e d e t e c t i o n system used. The i n t e r n a l standard i s added i n a known amount t o t h e sample p r i o r t o sample p r e p a r a t i o n and a n a l y s i s . A good i n t e r n a l standard i s a b l e t o e l i m i n a t e t h e b i a s caused by losses and compensates random e r r o r s d u r i n g e x t r a c t i o n o r a n a l y s i s ( r e f . 83). F i g . 2 shows a f l o w - c h a r t of s o l i d - l i q u i d e x t r a c t i o n procedures.

If

XAD-2 m a t e r i a l f o r t h e e x t r a c t i o n o f n e u t r a l and b a s i c drugs i s used i t can be renounced a t t h e d e p r o t e i n a t i o n s t e p ( r e f . 80). The p u r i f i c a t i o n s t e p can a l s o be performed a f t e r e l u t i o n o f t h e drugs from t h e s o l i d sorbent u s i n g l i q u i d - l i q u i d e x t r a c t i o n .

2-3

COLUMN-SWITCHING On-line sample preparation using column-switching has been applied to plasma, serum and urine samples and is discussed in detail i n Volume I. Blood sample analysis requires a preceeding purification step and is basically equal to analysis in plasma, serum or urine. Column-switching techniques for cyclosporine blood samples are described in part 3 . 2 . 3 of this chapter.

3. 3.1

BlOOD SAMPLE PREPARATION AND HPLC ANALYSIS OF SandimmunR (CYLOSPORINE) INTRODUCTION SandimmunR (Cyclosporine A, cyclosporine, Sandoz OL 27-400 N) is an immunosuppressive agent and i t s application after organ transplantation has proved to be of great value (refs. 84-85). Due to its narrow therapeutic range and its pharmacokinetic properties, blood level monitoring is mandatory. (ref. 86). Simultaneous measurement of the parent compound and the cyclosporine metabolites in blood by HPLC is of great clinical relevance, since the

97

commercially available and commonly used monoclonal radioimmuno assay (RIA) kits (Sandoz) (ref. 86) measure the parent compound or all metabolites to an mostly unknown extent. With HPLC it is possible to determine the metabolites and to quantify each of the metabolites separately. This will be of special value if one or more of the metabolites prove to be responsible for the cyclosporine adverse effects especially nephrotoxicity. Cyclosporine is a neutral, lipophilic and cyclic undecapeptide with a molecular weight of 1202.6. All its amino acids are S-configurated except D-alanine in position 8 (Fig. 1). Amino acids in positions 1, 3, 4, 6, 9, 10, and 11 are N-methylated. The amino acid in position 1 is a O-hydroxilated, N-methylated and unsaturated C9-amino acid. The tertiary structure of cyclosporine is an antiparallel R-pleated sheet conformation. Its partition coefficient octanol/water is 120/1. The cyclosporine molecule lacks of chromophoric substituents, making UV-detection more unspecific and demanding more extensive extraction procedures. The molar absorption coefficient at the wave-length maximum (195 nm) is 66 000 l/mol x cm. It shows good solubility in alcohols, ether, acetone and chlorinated hydrocarbons and poor solubi1 i ty in water and saturated hydrocarbons (refs. 88-91). Cyclosporine is metabolized by microsomal cytochrome P450 (ref. 92) in the liver to more than 30 metabolites (ref. 93). The structures of the metabolites 1, 8, 9, 10, 13, 16, 17, 18, 21, 25, 26 (refs. 94 and 95), 203-218 (ref. 96) and two aldehyde metabolites (ref. 97) have been elucidated. All metabolites retain the cyclic undecapeptide structure and prove to be more hydrophilic than the parent compound. The reactions involved in cyclosporine degradation are demethylation, hydroxilation, oxidation and cyclization (Table 11). Choice of the bioloaical matrix (ref. 98) For routine drug monitoring cyclosporine is usually measured in blood. However, the question of the biological matrix is still under discussion. 58% of cyclosporine are bound to the erythrocytes in blood, 10 to 20% to the lymphocytes. In plasma cyclosporine is bound to lipoproteins, preferentially those of high and low density (refs. 99-103). The free fraction is 1-1.5% at 37OC (ref. 104). The distribution between blood and plasma is temperature dependent and is lowered from 37OC to room temperature (refs. 99, 105-108). Binding of cyclosporine to the lipoproteins i s also temperature dependent being highest at body temperature and decreasing linearly with lower temperature. The cyclosporine metabolites 1 and 17 are associated with the erythrocytes

98 (>go%) ( r e f s . 109-111). The r e l a t i v e d i s t r i b u t i o n i n b l o o d i s constant u n t i l c y c l o s p o r i n e c o n c e n t r a t i o n s > 1000 ~ / 1 . Furthermore t h e r e l a t i o n between c o n c e n t r a t i o n i n b l o o d and plasma v a r i e s s i g n i f i c a n t l y w i t h t h e hematocrit ( r e f s . 112 and 113). The reasons f o r choosing blood as t h e b i o l o g i c a l m a t r i x are: 1. There a r e no t e c h n i c a l problems because o f t h e temperature dependent

d i s t r i b u t i o n between e r y t h r o c y t e s and plasma. 2. Measurement i s independent o f t h e hematocrit.

3. Plasma l e v e l s a r e considerably increased i n hemolysed b l o o d samples. The choice o f t h e a n t i c o a g u l a n t used f o r c y c l o s p o r i n e blood samples proved t o be o f importance. I n r o u t i n e h e p a r i n i z e d specimens s t o r e d > 1 days c o n t a i n small blood c l o t s .

Since c y c l o s p o r i n e i s bound t o a g r e a t

percentage t o t h e corpuscular blood components c l o t t i n g causes a decrease i n t h e c o n c e n t r a t i o n measured and t h e c l o t s p l u g t h e e x t r a c t i o n columns i n solid-phase e x t r a c t i o n procedures ( r e f s . 4, 98, 114-116). The

methods

developed

for

the

quantitative

determination

of

cyclosporine and i t s m e t a b o l i t e s i n blood cover almost t h e whole spectrum o f blood sample p r e p a r a t i o n s t r a t e g i e s .

99

I

CH2

I

AA8 Fig. 3

AA2

AAll AA1 -

AAlO

CH3

CH3

I

I

AA 7

CH-OH

I

AA6 -

I CH2

CH3

I

I

AA5

Structural formula of cyclosporine.

AA4

100

TABLE I 1

Structures o f the cyclosporine (Cs) metabolites, hitherto characterized (refs. 94-96) with their molecular weights.

I R

R1

R2

H

CH3

1 8

OH

CH3

CH3 CH3

OH

CH20H

CH3

9 10 13

OH

CH3

H

H

OH

OH

CH3

CH3

OH

H

16 17

OH

CH3

CH3

H

H H

CH20H CH20H

CH3

H

H

1234.64 1218.64

CH3

H

H AA1:cyclization

1218.64

H H

CH3 CH20H

H

H

H

1188.62

25

H

H

H

1204.64

26

OH

CH20H

CH3

H

H AA1:cyclization

1234.62

203-218

H

COOH

CH3

H

H

1232.62

Metabolite cs

18 21

modifications weight

R3

R4

H H

H

1202.64

H

1218.64 1234.64

hydroxyl ated and N-demethyl ated derivative of cs OH

1220.62 1234.64 1204.62

TABLE 111 Characteristics of various HPLC procedures for quantitative determination of cyclosporine

Extraction Ref. matrix preparation

extraction

117 plasma + water urine

diethyl ether

118 plasma

cv

HPLC clean-up recovery column elution det.-limit (pg/1 1

-

76+5% 10455%

RP8

gradient

extraction identical with ref. 117

n.r.

RP8

isocratic

5

119 blood + distilled plasma water

diethyl ether

hexane

74% 49%

RP18

isocratic

25

9.2

120 serum

C18 cartridge (Sep-Pak, Waters)

water/ 90+10% methanol

RP18

isocratic

n.r.

n.r.

step gradient

25

3

RP18

gradient

50

77.356%

TMS

isocratic

100

n.r.

83-99%

RP8 isocratic ultrasphere

31

3.6-

methanol

121 plasma heat (55OC), blood freeze + thaw

column-switching

122 serum

CN cartridge (Baker)

123 plasma

Clin Elut

water/ 50-70% methanol

cartridge (Fisher)

124 plasma acidification diethyl ether blood (HC1)

91.9+0.9% RP8, RP18

NaOH

20

comments

%

n.r.

4.4 derivatization o f cyclosporine with 2naphthyleneselenylchloride

9.314.1

6.0

TABLE I I I 125

blood serum

Tris-buffer pH 9.8

126/ plasma 127 blood

81 blood

c

(conti nued)

0 N

diethyl ether/ aceton- 34.7% C N cartridge nitrile/ (Baker) water, hexane

acetonitri le/water, column-switching freeze + thaw acetonitrile acetonitrile

128

blood freeze+thaw diethyl ether plasma buffer pH 10

129

serum

phosphoric colum-switching acid in acetonitri le

130

blood

freeze+thraw charcoal slurry acetonitrile ethyl acetate

131 blood,

plasma

132

blood

-

diethyl ether

10% isoC18 cartridge propanol in (Baker Bond) acetonitri le

RP8

isocratic

25

21

RP8, RP18

columnswitching

5 15

0.511.1

hexane, 90%+5% Dowex ion exchange resin

RP18

isocratic

25

acidifi- 74% cation, 85% hexane

RP18

isocratic

25

n.r.

RP8, RP18

columnswitching

n.r.

RP18

isocratic

50

CN

isocratic 100

6.04

isocratic

n.r.

hexane

100%

80%

alkalized 96+6% acidi fi ed met h ano 1 70%

methanol

86+108% C N

50

automated sample preparation

0.3-

8.0

7.0

2.512.5

8.6

modification of refs. 126, 127

TABLE 111

(continued)

110 blood acetonitrile/ CN cartridie serum, water (Bond Elut ) plasma (30/70,v/v)

acetonitrile/ acetic acid

90% 98%

CN

isocratic

133 blood

acetonitrile/ C18cartridfle di methy 1 (Bond Elut ) sulfoxide

acetoninitrile water, hexane

75-

CN

i socrati c
6.46.6

134 blood

--

acidification, hexane

n.r.

RP18

isocratic

n.r. modification o f ref. 119

135 blood

acetonitrile columnswitching

acetonitrile/ water

75~3% Ultrfl- columnpore switching RPSC (Altex) RP18, 3 vm

136 blood

HC1

diethyl ether

NAOH

137 blood

HCl

diethyl ether

heptane ~ 9 0 %

138 blood

acetonitrile

139 blood

freeze+thaw

diethyl ether

column-switching diethyl ether

hexane

80% ultrahere

15

n.r.

2

RP8

isocratic

20

RP8, 3 Ilm

isocratic

10

98.4100.2%

CN, TMS

columnswitching

5

108%

RP6, RP18

columnswitching

25

n.r.

2.6- determination of a 6.5 cell-bound metabolite

0.16.2

1.8

4.5

3.9 ion-pair chromato5.7 graphy (ammonium su 1 fate) 0.71 .8

5.7- extraction equal to 6.3 ref. 119, microbore analytical column c-l

0

w

c 0

TABLE 111

(continued)

140, blood 141

acetonitrile/ C18 cartridge methanol(9/1) (Bond Elut )

142 blood

HC1

76 blood

77 serum

145 blood, pl asma, bile

hexane, n.r. silica gel cartridge

25

4.9

88-90% RP8 isocratic methyl-t-butyl- NaOH, ether heptane mi crobore

1.5

4.8- small sample volume 5.9 (0.2-0.5 ml)

hexane, 98-104% aceton i tri 1 e/ water

RP18, 3 Pm

isocratic

20

4.5- extraction with ad7.8 vanced automatic sample unit AASP (Varian)

95-108%

CN

isocratic

15

6.6- modification of 6.9 ref. 135

75-83.9

RP18

isocratic

50

18.45.6

hexane, 87% aceton i tri 1 el water

CN,

isocratic

20

NaOH

RP18 3 Clm

gradient

22

C18 cartridge (Bond Elut ) HC1

diethyl ether

acetonitrile C8 cartridge (Analytichem)

HC1

RP1

isocratic

acetonitrile/ C8 cartridRe dimethyl(Bond Elut ) su 1 foxi de

143 blood 144 plasma, bile, urine

P

diethyl ether

NaOH

+7.7%

>96%

3 ccm

3.8- extraction with ad12.5 vanced automatic sample unit AASP (Varian), normal phase chromatography 5.6

determination of metabolite 1, 17, 18, 21

TABLE

II I

(conti nued)

I.46 blood,

plasma

147 blood

148 blood

HC1

diethyl ether

45~2% silica cartridge (Sep Pak, Waters), acetylacetate/ hexane

RP18

isocratic

1020

6

diethyl ether

heptane, 70% NaOH , hexane

RP8, 3 Pm

isocratic

25

5.311.5

acetoni- 47trile/ 95% water

CN isocratic 15a1 ternat i vely 25 RP 8, silica gel semi-preparative isolation of metabolites

acetoni- 73trile/ 85% water, hexane

RP8

acetonitrile/ CN cartridRe water (30/70) (Bond Elut )

149 blood, acetonitrile/ C8 extraction 150 bile, water(30/70) columns 151 urine

n.r.: not reported, RP: reversed phase.

gradient

25

7.1 determination of 9.6 metabolite 1 , 8, 13, 17, 18, 21, 25, 26, 203-218 and 1 yet unidentified metabolite

5.6 12.6

determination o f metabolite 1, 8, 9, 10, 13, 16, 17, 18, 21, 25, 26, 203-218 and 7 yet unidentified metabolites

106

BLOOD SAMPLE PREPARATION FOR SANDIMMUN~ (CYCLOSPORINE) MEASUREMENT 3.2 3.2.1 LIQUID-LIQUID EXTRACTION PROCEDURES A l l methods published u n t i l now use

c y c l o s p o r i n e C o r 0 as i n t e r n a l

standard. The e x t r a c t i o n procedures c o n s i s t o f f o u r steps:

1. hemolysis and d e p r o t e i n a t i o n , 2. e x t r a c t i o n o f cyclosporine, 3. sample p u r i f i c a t i o n , 4. volume r e d u c t i o n and t r a n s f e r i n t o t h e m o b i l e phase Hemolysis was achieved by r a p i d thawing and f r e e z i n g ( r e f . adding d i s t i l l e d water ( r e f . c h l o r i c a c i d (137).

119), a c e t o n i t r i l e ( r e f .

129) o r by

81) and hydro-

I n r o u t i n e a n a l y s i s c y c l o s p o r i n e was e x t r a c t e d from b l o o d by d i e t h y l methyl-t-butyl ether (ref. e t h e r ( r e f s . 117,119,124,127,128,136-138),

142) and a c e t o n i t r i l e ( r e f . 81). The advantage o f m e t h y l - t - b u t y l e t h e r over d i e t h y l e t h e r a r e i t s r e s i s t a n c e t o peroxide f o r m a t i o n and c l e a n e r e x t r a c t s than obtained by d i e t h y l e t h e r e x t r a c t i o n ( r e f . 142). The use of

acetonitrile

combines

s o l v e n t and i t s p r o t e i n

i t s p r o p e r t i e s as

an e f f e c t i v e e x t r a c t i o n

p r e c e p i t a t i n g potency

(ref.

81). Since t h e

e x t r a c t s c o n t a i n i n t e r f e r i n g l i p o p h i l i c m a t e r i a l and a c i d i c , b a s i c and i o n i c contamination ( r e f .

81) which may cause damage t o t h e column,

p u r i f i c a t i o n steps are required. P u r i f i c a t i o n was achieved by washing t h e sample w i t h hexane o r heptane ( r e f s . 119,128,134,137,139,142)w i t h a c i d i c and basic s o l u t i o n s ( r e f s . 124,128,131,134,142) o r by adding i o n exchange r e s i n s ( r e f . 81). A f t e r e v a p o r a t i o n o f t h e p u r i f i e d l a y e r and resuspension i n t h e mobile phase, some methods use a second p u r i f i c a t i o n s t e p by e x t r a c t i n g i n t e r f e r i n g substances w i t h a f i n a l wash ( r e f s .

hexane o r heptane

137, 142). Back e x t r a c t i o n o f c y c l o s p o r i n e from an aqueous

phase by changing pH i s n o t p o s s i b l e because o f i t s chemical p r o p e r t i e s . Thus t h e organic l a y e r c o n t a i n i n g c y c l o s p o r i n e i s washed by b a s i c and a c i d i c s o l u t i o n s and t h e aqueous l a y e r has t o be discarded i n e i t h e r case. G f e l l e r e t . a l . ( r e f . 118) used a d e r i v a t i z a t i o n o f c y c l o s p o r i n e w i t h 2-naphthylselenylchloride t o improve t h e d e t e c t i o n l i m i t . The method o f Sawchuck and C a r t i e r ( r e f .

119) i n t r o d u c e d a hexane

washing s t e p i n t o c y c l o s p o r i n e a n a l y s i s and many l a t e r p u b l i s h e d l i q u i d l i q u i d e x t r a c t i o n methods used a m o d i f i c a t i o n o f t h i s e x t r a c t i o n p r o cedure ( r e f s . 128,137,139,142). Blood, d i s t i l l e d water and t h e i n t e r n a l standard Cyclosporine D were g i v e n i n t o a c e n t r i f u g e tube. D i e t h y l e t h e r was added and t h e sample shaken and c e n t r i f u g e d . The aqueous phase was discarded and t h e organic l a y e r was evaporated. The sample was taken up

107 i n methanol and was washed w i t h hexane t w i c e . The hexane l a y e r s were removed, t h e aqueous l a y e r was b a s i f i e d w i t h NaOH and c y c l o s p o r i n e was e x t r a c t e d by d i e t h y l e t h e r . The d i e t h y l e t h e r phase was evaporated and t h e re ma ining m a t e r i a l s were r e c o n s t i t u t e d w i t h t h e m o b i l e phase. Most

of

these

extraction

Cy c los porin e D o r C.

procedures

use

an

internal

st andard:

Cyclosporine D i s cyclosporine w i t h v a l i n

and

c y c l o s p o r i n e C w i t h t h r e o n i n e as amino a c i d 2 (F ig. 3 ) . These c y c l o s p o r i n e d e r i v a t i v e s r e p r e s e n t o n l y a s m a l l m o d i f i c a t i o n o f t h e whole molecule. They

have

distribution

coefficients

in

organic

solvents

equal

to

c y c l o s p o r i n e and almost t h e same UV-absorbing p r o p e r t i e s . The use of t h e s e i n t e r n a l s t a ndar d s f o r t h e q u a n t i f i c a t i o n o f c y c l o s p o r i n e m e t a b o l i t e s i s critical

(ref.

152).

The b e h a v i o r d u r i n g e x t r a c t i o n

i s considerably

d i f f e r e n t f rom t h e m e t a b o l i t e s as shwon i n b i l e i n r e f . 151 and Table V . 3.2.2 SOLID-LIQUID EXTRACTION PROCEDURES U n t i l now a l l column e x t r a c t i o n

methods described f o r c y c l o s p o r i n e i n -

cl uded 5 s t e ps : 1. Hemolysis o f t h e c o r p u s c u l a r b l o o d i n g r e d i e n t s and d e p r o t e i n a t i o n , 2. sample l o a d i n g on t h e e x t r a c t i o n column, 3. sample p u r i f i c a t i o n , 4. e l u t i o n o f c y c l o s p o r i n e and i t s m e t a b o l i t e s f rom t h e e x t r a c t i o n column,

5 . volume r e d u c t i o n f o r HPLC a n a l y s i s . Yee e t a l . ( r e f . 122) used a p r o t e i n p r e c i p i t a t i o n s t e p w i t h a c e t o n i t r i l e c o n t a i n i n g t h e i n t e r n a l s t a n d a r d C y c l o s p o r i n e D. The sample was t h e n p u l l e d by vacuum through a prepacked d i s p o s a b l e cyanopropyl column, b e i n g washed w i t h a c e t o n i t r i l e and w a t e r . The column was washed w i t h methanol/ wat e r 40/60 ( v / v ) and c y c l o s p o r i n e was e l u t e d u s i n g methanol. Kates e t a l . ( r e f . 125) combined a d i e t h y l e t h e r e x t r a c t i o n w i t h p u r i f i c a t i o n on

prepacked d i s p o s a b l e cyano columns.

Blood samples were

a d j u s t e d a t pH 9.8 and e x t r a c t e d w i t h d i e t h y l e t h e r . D i e t h y l e t h e r was evaporated, t h e sample d i s s o l v e d i n methanol/water was d i l u t e d w i t h w a t e r and drawn t hro ug h t h e column w i t h water. The sample was subsequent ly cleaned by a c e t o n i t r i l e / w a t e r 25/75 ( v / v ) and hexane was t hen e l u t e d f r o m t h e column w i t h methanol. The method developed i n o u r l a b o r a t o r y

(refs.

149,150,151,

Fig.

4)

uses 3 m l g l a s e x t r a c t i o n columns f i l l e d w i t h 25-40 p RP 8 m a t e r i a l R (L iC hro pre p , Merck, Darmstadt, FRG). The i n t e r n a l st andard C y c l o s p o r i n e D was d i s s o l v e d

in

a c e t o n i t r i l e / w a t e r (pH 3.0) 50/50 ( v / v ) a t a concen-

108

tration of 10 @/ml. 25 pl of the internal standard solution were pipetted into a 10 ml centrifuge tube. Subsequently 1 ml blood and 2.1 ml acetonitrile/water (pH 3.0) (30/70 v/v) were added. Each sample was vortexed for 20 s and centrifuged for 5 min at 2 500 rpm. The supernatant was pulled by vacuum through the extraction columns. The extraction columns were previously primed with 3 ml acetonitrile and 3 ml water. The samples were washed with 3.2 ml acetonitrile /water (pH 3.0) (20/80 V/V) and with 0.5 ml hexane. The column was dried by sucking air through it for 1 min. To elute cyclosporine and its metabolites the extraction column was set into a diethyl ether cleaned 10 ml centrifuge tube and 2 ml dichloromethane was centrifuged through the extraction columns (700 rpm, 5 min). Dichloromethane was evaporated and the remaining materials were taken up in 300 pl acetonitrile/ water (pH 3.0) (50/50 v/v). 500 d hexane were added and the sample was vortexed for 10 s. Phases were separated and 100 ml of the aqueous phase were injected into the HPLC system. This extraction procedure is a modification of the method pub1 ished by Lensmeyer and Fields (ref. 110). The first step of the extraction procedure consists of adding a mixture of acidified water (pH 3.0) /acetonitrile (30/70 v/v) resulting in a final acetonitrile concentration of 20% in the sample. According to ref. 110 gross protein precipitation occurs at a final acetonitrile concentration of more than 21%. At the acetonitrile concentrations reached in the sample blood cells are hemolysed and some protein blood components precipitate. The recovery is considerably lower at a higher pH of the dilution mixture. The recovery drops to about 20% when gross protein precipitation occurs due to plugged extraction columns. Critical conditions for gross protein precipitation are high temperatures over 25OC as reached when centrifuging the sample in a warm centrifuge. Another reason for a decreased recovery is the extraction of deep frozen or samples stored at +4OC for more than 1 week. The first step also adjusts the sample to the conditions required for column extraction. After centrifugation the supernatant which has a clear red color is given onto the extraction columns. We chose no commercially available disposable prepacked columns but refillable glas columns with removable teflon frits for the following reasons: 1. To reduce costs of external column extraction procedures the ex-

traction columns are reused. The more often they are reused the more reproducibility and recovery decrease and the chance of loosing a

109 sample because o f a plugged column increases. A f t e r a n a l y s i s t h e s o l i d phase o f t h e g l a s s columns i s removed and t h e f r i t s reusable f o r a t l e a s t t h r e e times a r e cleaned by u l t r a - s o u n d i n a c e t o n i t r i l e . 2. One o f t h e main problems o f c y c l o s p o r i n e a n a l y s i s a r e i n t e r f e r i n g m a t e r i a l s l i k e p l a s t i c s o f t e n e r s which have s i m i l a r chromatographic and s p e c t r a l p r o p e r t i e s l i k e c y c l o s p o r i n e i t s e l f . I t has been r e p o r t e d t h a t i n t e r f e r i n g m a t e r i a l can be leached from t h e e x t r a c t i o n columns. Glass i s an i n e r t m a t e r i a l . A ' d i g i t a l chromatography' o f c y c l o s p o r i n e on t h e e x t r a c t i o n columns i s

n o t p o s s i b l e r e s u l t i n g i n v a r i a b l e r e c o v e r i e s o f 7 0 4 5 % i n o u r system. I n our method t h i s v a r i a b i l i t y can be compensated by u s i n g an i n t e r n a l standard (Table I V ) . The columns a r e f i l l e d w i t h 100 mg RP 8 s o l i d phase m a t e r i a l . V a r i a t i o n o f t h e packing volume up t o 50% does n o t i n f l u e n c e recovery. pH adjustment o f t h e sample t o an a c i d pH increases r e t e n t i o n on t h e columns e s p e c i a l l y o f t h e c a r b o x y l a t e d m e t a b o l i t e 203-218.

After

l o a d i n g c y c l o s p o r i n e and i t s m e t a b o l i t e s o n t o t h e e x t r a c t i o n columns, t h e

(80/20 v / v ) m i x t u r e , decreasing t h e amount o f p o t e n t i a l l y i n t e r f e r i n g m a t e r i a l . During t h i s s t e p t h e recovery i s n o t reduced when t h e water i s a c i d i f i e d . The n e x t sample i s washed w i t h an a c e t o n i t r i l e / w a t e r (pH 3.0)

step, washing t h e column w i t h hexane i s t o remove l i p o h i l i c i m p u r i t i e s . c y c l o s p o r i n e and i t s m e t a b o l i t e s a r e almost i n s o l u b l e i n hexane. Up t o t h i s s t e p t h e s o l v e n t s are sucked through t h e column by vacuum. To e l u t e t h e compounds o f i n t e r e s t from t h e e x t r a c t i o n column t h e columns a r e s e t i n c e n t r i f u g e tubes.

The e l u e n t dichloromethane i s c e n t r i f u g e d t h r o u g h

t h e e x t r a c t i o n columns and t h e e l u a t e c o n t a i n i n g c y c l o s p o r i n e and i t s metabolites

i s collected a t

the

bottom o f t h e c e n t r i f u g e

tube.

In

c o n t r a s t t o o t h e r e l u e n t s l i k e methanol o r a c e t o n i t r i l e dichloromethane can f a s t e r be evaporated, The amount o f c o e l u t e d i n t e r f e r i n g m a t e r i a l i s equal t o an e l u t i o n by o t h e r s o l v e n t s . For evaporation o f dichloromethane an apparatus equipped w i t h g l a s s tubes f o r n i t r o g e n i n s u f f l a t i o n should be used, s i n c e p l a s t i c tubes a r e a p o t e n t i a l source o f p o l l u t i o n of t h e sample w i t h p l a s t i z i s e r s (Fig.

10). The f i n a l hexane wash used i n o u r

method removed i n t e r f e r i n g m a t e r i a l , stemming from t h e l a b o r a t o r y equipment. This step was n o t e s s e n t i a l b u t i t made e x t r a c t i o n more re1 i a b l e . With s l i g h t m o d i f i c a t i o n s t h e method c o u l d be adapted t o analyse u r i n e and b i l e samples. 1 m l u r i n e was p i p e t t e d i n t o a 10 m l c e n t r i f u g e t u b e and 300 pl a c e t o n i t r i l e were added. A f t e r c e n t r i f u g a t i o n t h e supernatant

110 was passed through t h e e x t r a c t i o n columns. 1 m l o f a b i l e sample and 2 m l a c e t o n i t r i l e / w a t e r (pH 3.0) acetonitrile/water

(pH 3.0)

discarded.

loading

After

(30/70 v/v) were washed w i t h 3 m l hexane. The phase was separated and t h e hexane l a y e r cyclosporine

and

its

metabolites

on

the

e x t r a c t i o n columns the e x t r a c t i o n procedure was continued as described f o r blood samples.

1 m l blood + 25 pl i n t e r n a l standard (containing 250 ng cyclosporine D) + 2.1 m l a c e t o n i t r i l e / w a t e r (pH 3.0) 30/70 (v/v)

e x t r a c t i o n column

+ 3.2 m l a c e t o n i t r i l e + 3.2 m l water (pH 3.0)

I I

shake 20 s (vortex-mixer) c e n t r i f u g e 5 min, 2500 rpm

I I

I

-

draw supernatant through t h e e x t r a c t i o n column

I

+ 3.2 m l a c e t o n i t r i l e / w a t e r (pH 3.0) (20/80 v/v) + 0.5 m l hexane d r y column by a i r stream

I

c e n t r i f u g e 2.0 m l dichloromethane through t h e column

I

remove column m a t e r i a l teflon f r i t s

I clean

I

evaporate e l u a t e a t 50°C under a stream o f n i t r o g e n

I

+ 0.3 m l a c e t o n i t r i l e / w a t e r (pH 3.0)

(50/50 v/v)

+ 0.5 m l hexane

I

shake 20 s (vortex-mixer) c e n t r i f u g e 2 min, 2500 rpm

I

i n j e c t 75 pl i n t o HPLC-system Fig. 4

Extraction o f cyclosporine and i t s metabolites from blood by s o l i d l i q u i d e x t r a c t i o n ( r e f s . 149, 150, 151)

111

Modifications for the extraction of urine samples 1 ml urine + 25 pl internal standard

+ 300

pl

acetonitrile

I

vortex mix (40s) centrifuge 2 min 2500 x g

I (extraction continued like blood) Modifications for the extraction o f bile samples 1 ml bile + 25 pl internal standard + 2.1 ml acetonitrile/water (pH 3.0) + 2 ml hexane

(30/70 v/v)

I

vortex mix (1 min) centrifuge 2 min 2500 x g

I discard hexane layer

suck acetonitrile/water phase through extraction columns

I (extraction continued like blood) Reproducibility, linearity and detection limit of the method used in our laboratory are listed in Table I V . TABLE I V

Calibration curve, detection limit and CV of the method described above Calibration curve range checked

blood bile urine

0-3

mg/l 0-6 mg/l 0-30 mg/l

r 1.o

0.989

0.996

detection limit 25 c9/1 50 @/1 50 d l

cv 6.3% 7.2% 12.3%

112

The CV includes the variation of the cyclosporine metabolites. The recovery in blood ranged from 72-85% with an average of 79.2%. The recoveries of the metabolites 8 , 26, 17 and the internal standard Cyclosporine D are shown i n table V. The cyclosporine metabolites, the parent compound and the internal standard differ i n their lipophilic properties. Thus, the possibility must be taken into account that the recoveries of these compounds are not identical during the extraction procedure. In the table it is shown that the recovery of the internal standard i s signi fi cantly (p
Recoveries of the cyclosporine metabolites and the internal standard Cyclosporine D from bile and urine samples urine

Metabolite Cyclosporine D Metabolite 8 Metabolite 26 Metabolite 17 AV (metabolites)

bile

Recovery % 2 SD 78

-+

18

n

Recovery

8

15.2

+

4.2

7

3 2

78.7 71.8 69.4 73.3

t 8.2

3 3

73.4 78.1

8.9

85.1 79.5

2 4.2

3

6.9

8

%

5 SD

-t 5.2 + 16.6 + 10.7

n

3 9

AV: average. Kabra et al. (ref. 76) described an automated external column extraction by using an advanced automated sample processing unit (AASP, Varian, Walnut Creek, CA, USA) online with the LC. Blood samples were preextracted with acetonitrile/dimethyl sulfoxide (94/4 v/v) and hexane. C8 extraction columns were found to give a good recovery and

precision (refs. 76 and 77). The columns were primed w i t h and t h e aqueous phase + 1 m l water f o l l o w e d a c e t o n i t r i l e / w a t e r (2/3 v / v )

(2/3 v/v)

by a c e t o n i t r i l e / w a t e r columns were

were passed through t h e columns.

loaded on t h e AASP

and purged w i t h

The

acetonitrile/water

(12/13 v / v ) . Wallemacq e t a l .

(ref.

77) a l s o described a c y c l o s p o r i n e e x t r a c t i o n

procedure u s i n g t h e AASP, combines

the

standard

a p p l i c a t e d t o serum samples.

reversed

phase

extraction

with

This method normal

phase

chromatography. With a m o d i f i c a t i o n i t i s p o s s i b l e t o e x t r a c t and analyse already

structurally

metabolites.

elucidated

and

u n t i 1 now unknown

The authors t e s t e d C18, C8, C2, 2-OH,

cyclosporine

CN and S i e x t r a c t i o n

column f o r t h e i r e x t r a c t i o n procedure summarized below and found C8 m a t e r i a l t o g i v e t h e b e s t r e s u l t s . The r e c o v e r i e s f o r these columns were

73% (C18), 70% (C2), 64% (CN), 64% (2-OH),

20% (SI) and 87% f o r C8. F o r

p r o t e i n p r e c i p i t a t i o n a c e t o n i t r i l e and t h e i n t e r n a l standard were added to

a

serum

sample.

The

sample

was

washed

with

hexane

and

the

a c e t o n i t r i l e / w a t e r phase was separated and d i l u t e d w i t h water i n t o t h e AASP

cartridges.

acetonitrile/water

Using

the

(33/67

v/v)

AASP

the

sample

are

purified

with

and hexane and d r i e d by sucking a i r

through t h e e x t r a c t i o n columns p r i o r t o i n j e c t i o n i n t o t h e HPLC system. To move i n t e r f e r i n g components of t h e b l o o d samples Moyer e t a l . ( r e f s . 140 and 141) developed a double c a r t r i d g e s o l i d phase e x t r a c t i o n procedure u s i n g two subsequent e x t r a c t i o n columns f i l l e d w i t h C18 bonded phase

and

silica

gel.

After

protein

precipitation

with

a c e t o n i t r i le/methanol ( 9 : l ) t h e supernatant i s r i n s e d through t h e C18 e x t r a c t i o n column by vacuum and i s washed w i t h waterhnethanol (70/30 v/v) and acetone/hexane

(1/99 v / v ) .

The c y c l o s p o r i n s were e l u t e d w i t h i s o -

propanol and e t h y l a c e t a t e ( 1 / 3 V / V ) and t h e e l u a t e was passed through a s i l i c a g e l e x t r a c t i o n column, which d i d n o t r e t a i n t h e c y c l o s p o r i n s b u t i n t e r f e r i n g m a t e r i a l s . A f t e r evaporation and r e c o n s t i t u t i o n i n t h e m o b i l e phase t h e sample was i n j e c t e d i n t o t h e HPLC. Another approach was t h e e x t r a c t i o n o f c y c l o s p o r i n e by charcoal r e s i n s ( r e f . 133). A f t e r hemolysis by f r e e z i n g and thawing and e x t r a c t i o n w i t h a c e t o n i t r i l e 5 m l aqueous s l u r r y c o n t a i n i n g 10 mg charcoal were p i p e t t e d t h e supernatant. A f t e r c e n t r i f u g a t i o n t h e aqueous to

a g i t a t i n g t h e m i x t u r e on p o r t i o n was decanted and

a shaker and t h e remaining

charcoal e x t r a c t e d w i t h e t h y l a c e t a t e . The e t h y l a c e t a t e was evaporated and t h e r e s i d u e r e c o n s t i t u t e d i n t h e mobile phase.

114

EXTRACTION AND ANALYSIS BY COLUMN-SWITCHING On-line column-switching techniques have been applied in most cases in the analysis of drugs in plasma or serum and urine (refs. 153 and 154). Blood sample clean-up with this technique has been developed to determine cyclosporine. On-1 i ne col umn-switchi ng techniques have the fol 1 owing advantages compared with liquid-liquid and solid-liquid extraction procedures (ref. 155) : 3.2.3

minimal loss of the material to be analysed, 2. short total analysis time, 3. highly selective and reproducible analysis because of fully automated sample preparation, 4. facilitated concentration in trace analysis. 1.

Column-switching techniques for purification and analysis of blood samples consist of the following four steps: 1. hemolysis, deproteinization and pH-adjustment, 2. sample purification on a precolumn,

3. separation on the analytical column, 4. column wash procedures and reequilibration of the system.

In contrast to plasma or serum blood contains more lipids and proteins, which cause column overloading and an increase in column back-pressure if the sample is injected directly requiring a short sample preparation prior to injection. Hemolysis and deproteinization is achieved by adding equal amounts of acetonitrile to the blood samples. This mixture is shaken and centrifuged and the supernatant containing the material to be analysed i s loaded on the extraction column inside the HPLC-system (refs. 121, 135, 138). On the pre-column the sample is purified by washing with non-eluting organic solvents or organic solvent/water mixtures. By combining front-cut and end-cut procedures (ref. 155) early and late eluted components are eliminated and only the relevant part of the chromatogram is switched to the analytical column (heart-cut) (ref. 155). Most column-switching blood sample clean-up techniques for cyclosporine use a heart-cut procedure (ref. 121, 135, 138). Most column-switching techniques described f o r cyclosporine analysis use no internal standard but an actual calibration curve for cyclosporine quantification. They show excel lent reproducibi 1 i ty , recovery and detection limit (Table 111).

115 The column-switching technique described by Nussbaumer e t a l .

(ref.

121) was a m o d i f i c a t i o n o f t h e column-switching technique proposed by E r n i e t a l . ( r e f . 155). A f t e r p r o t e i n p r e c i p i t a t i o n w i t h a c e t o n i t r i l e t h e sample was i n j e c t e d onto a 40 x 4.6 mm 30-40 p RP 8 column kept a t room temperature where i t was washed. P u r i f i c a t i o n and a n a l y s i s o f t h e sample was performed by a step g r a d i e n t u s i n g subsequently m e t h a n o l l w a t e r l acetonitri le, water/acetonitrile, tetrahydrofuran and methanol as eluents. A f t e r clean-up t h e c y c l o s p o r i n e c o n t a i n i n g f r a c t i o n was switched by forward f l u s h i n g t h e pre-column ( r e f , 156) t o t h e 150 x 4.6 mm 5 f l RP

18 a n a l y t i c a l column by h e a r t - c u t .

The a n a l y t i c a l

column was kept a t

70°C. The method described by Schran e t a l .

(ref.

127) and Smith e t a l .

( r e f . 126) i n c l u d e d an automated o n - l i n e l i q u i d - l i q u i d p u r i f i c a t i o n step. P r o t e i n p r e c i p i t a t i o n was achieved by adding acetoni t r i l e / w a t e r (97.5/2.5 v/v)

t o t h e blood sample. The supernatant was i n j e c t e d i n t o t h e m i c r o -

processor c o n t r o l l e d Technicon system (Technicon Instruments, Tarrytown, NY, USA), wherein t h e sample was washed a u t o m a t i c a l l y w i t h hexane and was i n j e c t e d o n t o a C8 column, which was kept a t 75°C. The h i g h temperature

of t h e e x t r a c t i o n column improves s e p a r a t i o n r e s u l t i n g i n s m a l l e r c u t times and l e s s i n t e r f e r i n g m a t e r i a l on t h e a n a l y t i c a l column. Cyclosporine was e l u t e d w i t h a c e t o n i t r i l e / w a t e r o n t o a C18 column u s i n g a h e a r t - c u t

55/45

procedure.

(v/v)

and was loaded

From t h e C18 column

c y c l o s p o r i n e was e l u t e d i s o c r a t i c a l l y by a c e t o n i t r i l e / w a t e r 75/25 ( v / v ) . The b a s i c s t r a t e g i e s of two l a t e r p u b l i s h e d methods ( r e f s . were s i m i l a r t o t h a t of

Nussbaumer e t a l .

(ref.

121).

135,138)

After protein

p r e c i p i t a t i o n w i t h a c e t o n i t r i l e samples were loaded on t h e e x t r a c t i o n columns and p u r i f i e d by a c e t o n i t r i l e / w a t e r .

During e l u t i o n from t h e

e x t r a c t i o n column t h e c y c l o s p o r i n e c o n t a i n i n g f r a c t i o n was switched t o t h e a n a l y t i c a l column by h e a r t - c u t .

The main d i f f e r e n c e t o t h e e a r l i e r

method was t h e use of a p r o t e i n s e p a r a t i o n column, 5 p , 75 x 4.6 mm and a subsequent 75 x 4.6 mm,

3 p octadecyl column as a n a l y t i c a l column ( r e f . 135) and 30 x 4 mm cyano-propyl 5 p precolumn combined w i t h a CLC-TMS, 5 p column (Shimadzu, Kyoto, Japan) which a l l o w lower oven temperatures (60

OC)

f o r b o t h a n a l y t i c a l and e x t r a c t i o n column and low

f l o w r a t e s t o o b t a i n a s u f f i c i e n t separation. The pre-columns were k e p t a t t h e same temperature as t h e a n a l y t i c a l columns. 3.3

CHROMATOGRAPHIC ANALYSIS

OF SANDIMMUN~ (CYCLOSPORINE) AND ITS

METABOLITES Chromatography o f c y c l o s p o r i n e and i t s m e t a b o l i t e s i s discussed i n d e t a i 1 i n t h i s chapter s i n c e chromatography of c y c l o s p o r i n e i s acquainted

116

with some interesting analytical problems that must also be considered in chromatographic analysis of other drugs. However, the measurement of cyclosporine metabolites is currently under discussion and will be of great impact when one or more of the metabolites will prove to be biologically active, which seems to be very likely. One of the chromatographic difficulties in HPLC analysis of cyclosporine is the characteristic peak broadening. It is the result of an interconversion of several cyclosporine conformers exhibiting different chromatographic characteristics. Chromatography at 4OC results in a splitting into single conformer peaks. High temperatures accelerate interconversion of the conformers resulting in sharper peaks, representing an average conformer composition (ref. 157). Further analytical conditions to overcome separation of cyclosporine conformers resulting in peak broadening are: 1. low pH at 3.0 - 5.0 of the mobile phase, composition o f the mobile phase, e.g. gradient elution, 3. using a more polar stationary phase for example C1, C2, or CN. 2.

Further positive effects of HPLC at elevated temperatures are (ref. 158) : 1. High temperatures decrease viscosity of the eluents allowing the use of sorbents with a smaller particle size and/ or pellicular sorbents, resulting in a greater column efficiency. 2. Increasing temperature accelerates sorption by increasing mass transfer and kinetic rates. This effect is of special benefit for chromatography of large or very lipophilic molecules, as which cyclosporine must be regarded.

Adverse effect of increased temperatures are: 1. Faster degradation of the stationary phase. 2. Acceleration of not desirable on-column reactions, as expressed by the dimensionless Damkohler number (ref. 159).

Both effects are of practical value for HPLC analysis of cyclosporine as discussed below. Assessment of cyclosporine analysis by supercritical fluid chromatography (SFC) will be the subject of further investigations. Some authors (ref. 119, 123) observed a fast degradation of their

117 a n a l y t i c a l columns w i t h i n 100-150 h. Other authors r e p o r t e d c o n s i d e r a b l y l o n g e r column l i f e times up t o 3000 i n j e c t e d samples ( r e f s . 137, 157). Reversed phase columns can be operated by temperatures up t o 100°C w i t h o u t degradation (personal communication, Merck, Darmstadt, FRG) the

beginning

of

a n a l y t i c a l columns

cyclosporine

measurement

in

our

laboratory

(C8) had a l s o a very reduced l i f e span.

.

At

our

The main

reasons turned o u t t o be t o o f a s t and f r e q u e n t h e a t i n g and c o o l i n g o f t h e column and t h e i n j e c t i o n o f p o o r l y p u r i f i e d samples. Thus o u r a n a l y t i c a l column was kept a t 75 "C w i t h a small f l o w o f 0.1 ml/min d u r i n g p e r i o d s w i t h o u t analysis.

The a n a l y t i c a l column was guarded by a

precolumn and

a f t e r each a n a l y s i s t h e column was f l u s h e d w i t h a c e t o n i t r i l e / w a t e r

3.0)

(pH

(90/10 v/v) t o remove peaks n o t b e i n g e l u t e d d u r i n g a n a l y s i s t i m e .

We found 2 peaks e l u t i n g a f t e r i n j e c t i n g b l o o d sample e x t r a c t s t h a t c o u l d n o t be detected a f t e r i n j e c t i o n o f c y c l o s p o r i n e standard s o l u t i o n s which were e l i m i n a t e d by t h e a c e t o n i t r i l e f l u s h .

Without t h e column f l u s h i n g

s t e p degradation o f t h e a n a l y t i c a l column occurred a f t e r a few hundred analyses r e s u l t i n g i n a proceeding decrease o f t h e t h e o r e t i c a l p l a t e s of t h e column and an i n c r e a s i n g back pressure o f t h e a n a l y t i c a l system. I n our system t h e r i s e i n pressure was caused by plugged i n l e t f r i t s of t h e precolumn and t h e a n a l y t i c a l column. I t disappeared a f t e r exchanging t h e metal f r i t s by t e f l o n f r i t s ( r e f s . 149 and 150). Lensmeyer e t a l . ( r e f s . 110,148) s e t a s i l i c a s a t u r a t i n g column between t h e pumps and t h e i n j e c t o r v a l v e t o minimize d i s s o l u t i o n o f t h e s i l i c a based sorbent i n t h e a n a l y t i c a l columns. Another approach t o extend column l i f e t i m e was t h e use o f a cyanopropyl

analytical

column which a l l o w s r e d u c t i o n of

the

column temperature t o 60°C as described above. U l t r a v i o l e t absorption from compounds n o t d e r i v e d from c y c l o s p o r i n e can produce peaks i n t h e chromatograms.

E s p e c i a l l y p l a s t i c s o f t e n e r s from

l a b o r a t o r y equipment and from r o t o r seals, valves and f r i t s i n s i d e t h e HPLC-system can d i s t u r b c y c l o s p o r i n e a n a l y s i s . I n our e x t r a c t i o n procedure o n l y d i e t h y l e t h e r cleaned c e n t r i f u g e tubes and i n j e c t o r v i a l s were used a f t e r t h e e l u t i o n step from t h e e x t r a c t i o n column and t h e f i n a l hexane wash removed remaining contaminations (2.2). I n our l a b o r a t o r y t h e f o l l o w i n g chromatographic system was developed and i s c u r r e n t l y i n use ( r e f . 150): R The column used a r e two s e q u e n t i a l 250 x 4.0 mm C8, 7 f l LiChrosorb f i l l e d manu-fix R c a r t r i d g e s guarded by a 50 x 4 mm pre-column f i l l e d w i t h t h e same m a t e r i a l ( a l l Merck, Darmstadt, FRG). Analysis were performed on an HP 1090 HPLC system (Hewlett Packard, Karlsruhe, FRG). Solvent A was water adjusted t o pH 3 w i t h phosphoric acid; s o l v e n t B a c e t o n i t r i l e . The

118

eluents were degassed by ultra-sound and vacuum prior to use and during run by helium insufflation. The flow rate was set at 1.4 ml/min, the detector wavelength at 205 nm and the oven temperature at 75OC. For elution three superimposed sequential linear gradients with increasing steepness resulting in an almost concave gradient were used. The gradient began at 43/57 (v/v) acetonitrile/water (pH 3.0) and increasing to acetonitrile/water (PH 3.0) 49/51 (v/v) until 20 min after injection, to acetonitrile/water (PH 3.0) 59/41 (v/v) until 35 min after injection and to acetonitrile/water (PH 3.0) 80/20 (v/v) until 45 min after injection to stay at this level for 5 min. The gradient was followed by a column clean-up step to remove late eluting lipophilic material otherwise occupying the binding sites of the column material resulting in decreasing efficiency of the column. The column was flushed with acetonitrile/water (pH 3.0) for 5 min and then reequilibrated to the start conditions with an eluent composition of acetonitrile/water (pH 3.0) 43/57 (v/v). Cycle time between two injections was 60 min. The stationary phase used for HPLC analysis ranged from silica gel to RP18 material. For our system C8 sorbent gave the best results. With more polar sorbents like C4, C 1 or CN a reasonable resolution especially of the metabolites 1 and 17 was not possible in our system. Wang et al. compared different columns of the same length and diameter filled with CN, phenyl and four different C18 packings under the same conditions analysing bile samples (ref. 160). In the study C18 columns gave the best results in resolving cyclosporine metabolites, especially 1 and 17. C8 columns were not included i n the study. Rocher et al. (ref. 161) found C8 columns superior to C18 columns in cyclosporine measurement. The two columns were tested under the same conditions and the concentrations measured with the C18 column were higher than those measured with the C8 column due to an interfering peak that was present in high concentration in patients with renal insufficiency and could be separated from the cyclosporine peak using a C8 column. Moyer et al. (refs. 140 and 141) proposed the use of a more polar phase to minimize cyclosporine band broadening. Unfortunately more polar columns are less effective in separating cyclosporine metabolites (ref. 160). One of the major problems in HPLC analysis of cyclosporine and its metabolites is the great number of metabolites. Until now more than 30 could be isolated in our laboratory (Fig. 5 ) . To increase the resolving power of our chromatographic system gradient elution and an elongated analytical column were used. The gradient consists of three linear gradients, since the cyclosporine metabolites detectable with our system

119

are eluted in three main groups (Metabolite 8, 9, 26; 13, 25; 203-218, 17, 1, 18, 21). Each of the linear gradients was designed to get the best resolution possible o f each metabolite group. Superposition o f the three linear gradient resulted in the concave gradient described (Fig. 6 ) . Mass spectrometric data of cyclosporine metabolites corresponding to Fig. 5, isolated from human bile or generated by human liver microsomes.

TABLE V I

metabol i te

molecular weight

f ormu 1 a Cs+20 cs + 0

1

H355

1234.4

2

17

1218.4

3 4

8

1234.6 1232.4

5

H410/420

6 7 8

203-218

9 10 11

H230

1216.4 1220.4 1217.4

c s + 2 0 CS + 2 0 - 2 H CS+ 0 - 2 H c s + 2 0 CS + 2 0

1232.4

CS + 2 0

1250.4

c s + 3 0

1264.4

CS + 4 0

1248.4

CS + 3 0 cs + 0 c s + 2 0

-

-

2 H

-

CH3

CH3

2 H 2 H 2 H

12

18

1218.4

13 14 15

H310 26

1234.6 1234.6 1394.2

c s + 2 0 Cs + 0 + glucoronic

1219.4

c s + 2 0

1250.4

c s + 3 0

H235

acid 16 17

-

18

1

1218.4

cs +

19

10

1234.4

20

1251.4

21

1248.4

CS + 3 0

1234.6 1188.4

c s + 2 0 cs

1203.4

cs +

0

1203.4

cs +

0

1219.4

c s + 2 0

1219.4

c s + 2 0

1235.4

c s + 3 0

1204.4

+

23 24 25 26 27

16 21 13

9

28 29

25

cs

CH3

-

CH3

0

c s + 2 0 c s + 4 0

22

-

0

-

2 H

-

-

CH3 CH3

CH3

CH3 CH3 CH3 CH3

120

26 -81 -

7

4 24 5 23 --

2---12 18

10 20 -

-

9 17 1115

28

27

3---14

I

0 Fig. 5

1

10

I

20

I

30

I

40

1

50

time (min)

Shows a chromatogram of a sample containing human microsomes at a concentration o f 3 mg protein/ml and cyclosporine. Cyclosporine and the microsomes were incubated for 2 h prior to extraction and analysis as described in the text (2.2, 3.3) and refs. 149151. Cyclosporine D was used as internal standard. The corresponding fractions were isolated by HPLC and further analysed by FABMS. The mass spectrometric data are listed in Table V I . The numbers of the metabolites in the Table correspond to the fraction numbers in the chromatogram. The nomenclature as proposed by refs. 94, 95 and 151 is used in the metabolite column o f Table V I . HPLC parameters Figure 5: Detector wavqlength: 205 nm, oven temperature: 75OC, plot attenuation: 2 , flow: 1.4 ml/min. Abbreviation Cs: cyclosporine

121

I

0

0

10

10

I

20

30

40

50 0

20

30

40

50 0

10

20

30

4 0 0 time (min)

fi, 10

20

30

40

50

time (min)

0

-.-Q

.

10

20

30

40

I

50

122

With the HPLC procedure described, 11 structurally elucidated as well as 7 not yet structurally known cyclosporine metabolites could be quantified and isolated from urine and bile of grafted patients (Fig. 7 ) . For peak identification the chromatogram was fractionated and the corresponding materi a1 was isolated by semi -preparative HPLC. Constituents of an isolated fraction were regarded to be identical with authentic cyclosporine metabolites if: 1. no peak with an equal retention time was found in blank samples of not cyclosporine treated patients, 2. the unspecific cyclosporine radioimmunoassay cross-reacted with the fraction in question, 3. the material in the fraction showed mass spectrometric properties implying the structure of a cyclosporine derivative, 4 . the isolated fraction and the authentic metabolite have the same retention time under the chromatographic conditions described above (Fig. 8)

J I

0

I

10

I

20

I

30

I

40

I

50 tima (min)

Fig. 7

Chromatogram of a urine extract of a liver grafted patient. The sample was extracted and analysed according to 2.2, 3.3, refs. 149-151. The HPLC system and parameters were the same as described in Fig. 6. The numbers indicate cyclosporine metabolites not yet structurally identfied using the nomenclature developed in our laboratory (ref. 151). These metabolites could be i s o lated by semi-preparative HPLC and their biological activity was evaluated. They can be quantitatively determined from the chromatograms using the internal standard cyclosporine D (Cs D). The corresponding FAB-MS data are listed in Table 6. Cs: cyclosporine. Arrow indicates injection.

123 1

fraction 21

L A . 1 10 20

I

30

0

(0

timo (min)

I 0

I

I

1

1

10

20

30

40

time (min)

Fig. 8

Chromatograms o f f r a c t i o n 21 ( t o p ) i s o l a t e d from a u r i n e e x t r a c t of a l i v e r g r a f t e d and c y c l o s p o r i n e t r e a t e d p a t i e n t and t h e a u t h e n t i c m e t a b o l i t e 16 (bottom), i s o l a t e d and i d e n t i f i e d as described i n r e f s . 94 and 95. The chromatographic c o n d i t i o n s and HPLC parameters were t h e same as described i n 3.3 and r e f . 150. The a n a l y s i s were s t o ped a f t e r 40 min. Arrow i n d i c a t e s i n j e c t i o n . Both peaks a r e e l u t e d from t h e column w i t h a r e t e n t i o n t i m e o f 33.7 min.

(i)

I n HPLC a n a l y s i s considered:

o f cyclosporine

the following

problems must

be

1. The l a r g e number o f c y c l o s p o r i n e m e t a b o l i t e s and t h e i r o n l y small s t r u c t u r a l d i fferences make a reasonable r e s o l u t i o n almost impossible. Figure 5 shows t h e m e t a b o l i t e s

that

c o u l d be i s o l a t e d by

semi-

p r e p a r a t i v e HPLC from a suspension o f human l i v e r microsomes (3 mg/ml p r o t e i n ) a f t e r i n c u b a t i o n w i t h 10 19 c y c l o s p o r i n e f o r 2 h. The numbers i n d i c a t e t h e peaks o r r e g i o n s i n t h e chromatogram where t h e c o r r e s ponding

metabolite

structural

data

as

could

be

isolated

elucidated

by

and

fast

are atom

assigned

to

bombardment

the mass

spectrometry (FAB-MS). These d a t a i n d i c a t e t h a t several peaks i n t h e chromatogram represent a conglomerate o f several m e t a b o l i t e s . AS

124 example t h e i s o l a t e d f r a c t i o n c o n t a i n i n g m e t a b o l i t e 9 i s shown i n F i g . 9. Although t h e peaks seem symmetric and pure a f t e r i n j e c t i n g t h e i s o l a t e d f r a c t i o n i n t o t h e HPLC, FAB-MS d e t e c t s t h r e e peaks r e p r e s e n t i n g d i f f e r e n t cyclosporine derivatives.

Fraction 8

HPLC

1 /

r

’“1 -

0

10

20

30

40

12181 11230

Qh

1

0 1000

.

1200

50 time (min)

- FAB - ms 0

1400 molacular weight

Fig. 9

The chromatogram (top) shows f r a c t i o n 8 c o n t a i n i n g m e t a b o l i t e 9 (nomenclature r e f . 94) , i s o l a t e d by semi -preparat ive HPLC from u r i n e o f a l i v e r g r a f t e d p a t i e n t and r e i n j e c t e d i n t o t h e HPLC system. The chromatographic c o n d i t i o n s and HPLC parameters a r e t h e same as described i n 3.3, Fig. 6, r e f s . 150 and 151. Although t h e peak i s symmetric and seems t o be caused by a monosubstance, t h e corresponding FAB-MS (negative mode) (bottom) a n a l y s i s showed a t l e a s t 3 d i f f e r e n t c y c l o s p o r i n e d e r i v a t i v e s ( r e f . 151). Arrow i n d i c a t e s i n j e c t i o n .

2. I t i s very l i k e l y t h a t t h e chromatographic c o n d i t i o n s (pH 3.0,

75OC)

change t h e c y c l o s p o r i n e m e t a b o l i t e p a t t e r n detected. Henricsson e t a l . ( r e f . 162) r e p o r t e d t h e i d e n t i f i c a t i o n o f a s u l f a t e d c y c l o s p o r i n e d e r i v a t i v e , i s o l a t e d by ion-exchange chromatography. Unter HPLC c o n d i t i o n s described above t h e b i n d i n g of s u l f a t e d o r glucuronated d e r i v a t i v e s can be hydrolyzed.

3. P l a s t i c m a t e r i a l leached from l a b o r a t o r y t o o l s have s i m i l a r s p e c t r a l properties

as

the cyclosporins

and s i m i l a r r e t e n t i o n times. F i g . 10

125 shows a chromatogram o f a b l o o d sample o f a l i v e r g r a f t e d p a t i e n t w i t h o u t c y c l o s p o r i n e therapy. The sample had c o n t a c t w i t h a p o l y p r o p y l e n t u b e o f t h e evaporation apparatus. Several peaks i n t h e range o f t h e c y c l o s p o r i n e m e t a b o l i t e s were detected. The chromatogram below i n t h e same Fig. shows a chromatogram o f p l a s t i c material.

t h e same r e - e x t r a c t e d

sample w i t h o u t

interfering

I n t e r f e r e n c e o f o t h e r drugs w i t h t h e HPLC assay has

been reported f o r sulfamethoxazole ( r e f . 163). 4. The q u a n t i f i c a t i o n o f c y c l o s p o r i n e m e t a b o l i t e s i s i n f l u e n c e d by t h e chromatograpic system used. A peak s e p a r a t i o n down t o t h e b a s e l i n e i s n o t p o s s i b l e f o r most o f t h e c y c l o s p o r i n e m e t a b o l i t e s . The peak area t h e r e f o r e depends on t h e i n t e g r a t i o n s o f t w a r e and i n t e g r a t i o n method used. As o u t l i n e d above most peaks do n o t represent a monosubstance. Using an HPLC system w i t h a g r e a t e r r e s o l v i n g power may r e s u l t i n two o r more s m a l l e r peaks ( r e f . 161). Therefore t h e r e p r o d u c i b i l i t y i s good f o r one HPLC system b u t comparison o f t h e m e t a b o l i t e concent r a t i o n s measured w i t h o t h e r l a b o r a t o r i e s i s d i f f i c u l t . Lensmeyer e t a l .

(ref.

148) compared t h r e e chromatographic systems

u s i n g a 80 x 4 mm 5 p o c t y l column, a 160x4 mm 5 p s i l i c a g e l column and a 250 x 4.6

mm cyano-propyl

column.

p r e f e r r e d and r u n w i t h water/ acidln-butylamine (600/390/20/0.16/ r e s u l t s i n t h e systems t e s t e d .

The cyano propyl-column

was

a c e t o n i t r i leltetrahydrofuranelacetic 0.1 v/v/v/v/v/) gave t h e b e s t

The chromatograms

show an incomplete

separation o f t h e m e t a b o l i t e s 1 and 18. Bowers e t a l .

( r e f . 145) simultaneously analysed c y c l o s p o r i n e and i t s

m e t a b o l i t e s on a 50x4.6 mm 3 p octadecyl column u s i n g an i s o c r a t i c g r a d i e n t w i t h waterlmethanollacetonitrile temperature o f 70°C.

(34120146

v/v/v/)

a t an oven

Incomplete s e p a r a t i o n o f t h e h y d r o p h i l i c group of

cyclosporine metabolites

was

achieved and t h e m e t a b o l i t e s

were

not

q u a n t i t i v e l y determined i n b i l e . Burckart e t a l . ( r e f . 164) e x t r a c t e d c y c l o s p o r i n e and i t s m e t a b o l i t e s from pooled b i l e u s i n g a m o d i f i e d e x t r a c t i o n procedure according t o Sawchuk e t a l . ( r e f . 111). c y c l o s p o r i n e and i t s m e t a b o l i t e s were e l u t e d from t h e a n a l y t i c a l column by an a c e t o n i t r i l e l w a t e r g r a d i e n t . Rosano e t a l .

(ref.

109) e x t r a c t e d c y c l o s p o r i n e and i t s m e t a b o l i t e s

from blood u s i n g a l i q u i d - l i q u i d e x t r a c t i o n procedure w i t h d i e t h y l e t h e r . Cyclosporine and i t s m e t a b o l i t e s 1, 17 and 21 were seperated i s o c r a t i c l y . Wallemacq e t a l .

( r e f s . 77 and 165) used a normal phase HPLC system

f o r a n a l y s i s o f c y c l o s p o r i n e and i t s m e t a b o l i t e s . A 3 p p a r t i c l e s i z e CN column

and

hexane:

isopropanol

(85115 v/v)

as m o b i l e phase were used

126 ( r e f s . 77 and 165). Column temperature was 50

OC.

separated by semi-preparative p r o p e r t i e s were evaluated.

their

I

0

I

0

HPLC

and

28 m e t a b o l i t e s c o u l d be

mass

spectrometric

,

10

20

30

40 50 time (min)

10

20

30

40

50 time(min)

Fig. 10 The chromatograms show t h e HPLC a n a l y s i s o f t h e same blood sample received from a l i v e r g r a f t e d p a t i e n t w i t h o u t c y c l o s p o r i n e therapy f o r 10 days. The e x t r a c t i o n procedure, t h e chromat o g r a p h i c c o n d i t i o n s and HPLC parameters were t h e same as described b e f o r e (2.2, 3.3, F i g . 6, r e f s . 149-151). During e x t r a c t i o n , t h e sample showed i n t h e chromatogram above had come i n t o c o n t a c t w i t h a p o l y p r o p y l e n t u b e o f t h e evaporation apparatus r e s u l t i n g i n contamination w i t h p l a s t i c m a t e r i a l . The arrows w i t h t h e numbers according t o Maurer's nomenclature ( r e f s . 94 and 95) i n d i c a t e t h e r e t e n t i o n times o f t h e c y c l o s p o r i n e metabolites. The chromatogram shows t h e a n a l y s i s o f t h e same blood sample w i t h o u t contaminating m a t e r i a l s . Both samples c o n t a i n t h e i n t e r n a l standard c y c l o s p o r i n e D (CsD). Arrow i n d i c a t e s i n j e c t ion. 4.

TROUBLE SHOOTING I N DEVELOPMENT OF BLOOD SAMPLE CLEAN-UP PROCEDURES Unexpected losses o f t h e compounds o f i n t e r e s t d u r i n g blood sample

clean-up can be caused by t h e f o l l o w i n g f a c t o r s ( r e f . 2 ) : 1. Chemical breakdown o r a d s o r p t i o n o f t h e compounds o f i n t e r e s t because of a t o o l o n g s t o r i n g p e r i o d o r inadequate s t o r i n g c o n d i t i o n s , 2. adsorption o f t h e drugs on m e t a l , g l a s s o r p l a s t i c surfaces of

1 aboratory equipment,

127 3. c o - p r e c i p i t a t i o n d u r i n g d e p r o t e i n i z a t i o n , 4. inadequate pH o f t h e b u f f e r s and s o l v e n t s used, 5 . low p a r t i t i o n c o e f f i c i e n t i n t h e o r g a n i c e x t r a c t i o n s o l v e n t , 6. i n s u f f i c i e n t adsorption on t h e s o l i d phase m a t e r i a l used, 7. chemical breakdown because o f chemical, b i o l o g i c a l , photochemical and/or thermal i n s t a b i l i t y , 8. c h e l a t i o n e.g. w i t h heavy metals o r i n t e r c a l a t i o n ,

9. l o s s d u r i n g t h e evaporation s t e p because o f v o l a t i l i t y o f t h e drug, 10. i n s o l u b l e r e s i d u e a f t e r evaporation, 11. i n s u f f i c i e n t d e r i v a t i z a t i o n because o f an incomplete r e a c t i o n , by-products and removal o f excess reagents Contamination o f t h e sample d u r i n g e x t r a c t i o n and a n a l y s i s can be caused by: 1. c o e x t r a c t i o n o f i n t e r f e r i n g m a t e r i a l such as 1 i p o p r o t e i n s and 1 ip i d s / l ip o i ds , 2. use o f contaminated s o l v e n t s o r s o l v e n t s o f a minor p u r i t y , 3. i n t e r f e r i n g residues i n glass-ware and l a b o r a t o r y t o o l s , 4. leached p l a s t i c s o f t e n e r s from l a b o r a t o r y t o o l s and e x t r a c t i o n columns, 5. unadequately primed s o l i d phase column m a t e r i a l , 6. contaminated i n j e c t o r needle o f t h e i n j e c t i o n system, 7. s o l u b i l i s e d p l a s t i c m a t e r i a l from t h e v i a l s and t h e v i a l caps o f t h e i n j e c t i o n system, 8. s o l u b i l i s e d p l a s t i c s o f t e n e r s from t h e valves, r o t o r s e a l s and c a p i l l a r i e s o f t h e HPLC-system. REFERENCES 1.

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