- Email: [email protected]
A rapid procedure for the isolation of drugs and drug metabolites from plasma
CLINICA
CCA
CHIMICA
381
.4CTA
4714
A RAPID
PROCEDURE
~IET~~3OLIT~S
FROM
FOR
THE
ISOLATION
01;
DRUGS
AND
DRUG
PLASMA
SUMMARl
A rapid procedure for the extraction of neutral and basic drugs and drug metabolites with isopropanol from diluted plasma saturated with potassium carbonate has been developed. The isopropanol extract is suitable for analysis by thin layer or gas chromatography. Recovery experiments (radioactivity) with phenobarbital and caffeine
gave
IOO +
5%
recovery.
The absence
of emulsions
contributes
to the high
recovery.
INTRODUCTION
Many procedures for isolating neutral and basic drugs and drug metabolites from plasma or serum have been published. Almost invariably, the problem of emulsion formation is ignored or centrifugation is recommended. When normal plasma or serum is analysed, centrifugation will usually keep emulsion formation within acceptable limits. In clinical studies, however, the chemist is concerned more often than not with abnormal plasma, and intractable emulsions which do not respond to centrifugation or other laboratory manipulations are frequently obtained. Reliable quantitative data are unobtainable under these circumstances. This di~culty has been resolved by applying to plasma the salting-out procedure developed by Sunshine and co-workers1 for the rapid isolation of drugs and drug metabolites from urine. Diluted plasma, to which a small volume of isopropanol has been added, is saturated with potassium carbonate. IJpon centrifugation, the isopropanol layer containing drugsand drug metabolites separates as the upper phase. Proteins precipitated by the isopropanol and potassium carbonate are found at the interface. The isopropanol extract can be analyzed directly, or after transfer to methanol solution, by thin layer chromatography (TLC) ; it is possible to carry out preliminary screening for drug metabolites on large numbers of samples by TLC if this is desired. After conversion to suitable derivatives, compounds in the isopropanol fraction can Clin. Chirn. Acta, 37 (1972j
381-3X6
be analyzed by gas chromatography (GC), and gas chronlatograpll~~nl~~~s spectrometry (GCMS). A gas chromatograph-mass spectrometerPcomputer instrument (GC MSPCOM) is particularly useful for tllis work. Gas chrornatograpllic analysis provides a quantitative analysis as well as tentative identification of the drugs and drug metabolites present in plasma. X11 structural assignments, howcvcr, shnuld be con firmed by mass spectromctric
analysis.
EXPERIMENTAL
Heparinized
rat blood was obtained
by cardiac puncture
with pentobarbital. The plasma was separated immediately stored at - 13” until analyzed. Isopropanol, methanol and iso-octane were Nanogradc Chemical Co.). Anhydrous
potassium
carbonate
(granular)
was obtained
from rats anesthetized by- centrifugation quality
and
(Mallinckrodt
from Fisher Chemical
co. Isolatiol2 f~fom jdasma One ml of plasma was transferred to a x5-ml screw-capped centrifuge tube and diluted with 5 ml of glass-distilled water. One ml of isopropanol \vas added, followed by 6 g of anhydrous granular potassium carbonate. The contents of the tube were rapidly and thoroughly mixed by inversion; the solution was agitated in a Vortex mixer for zo set and then allowed to stand for 5 min. The phases separated during this time and the separation was completed by centrifugation for 5 min. The isopropanol layer (upper phase, 0.9 ml) was transferred wit11 a Pasteur pipette to a small scrcwcapped (Teflon-lined) tube (12 mm r: xoo mm). Tlie extraction was repeated twice by adding I ml of isopropanol to the potassium carbonate solution, mixing thoroughly with a \Tortex mixer and centrifuging for 5 min. The combined isopropanol extracts (approximately 3 ml) containing drugs and drug metabolites was evaporated ivith the aid of a nitrogen stream. The residue was dissolved in 0.5 ml of methanol for subsequent analysis by TLC or GC methods. Thin
layer chromatograf$ An aliquot (50-100 ~1) of the methanol solution (0.5 ml) was used for analysis by TLC. The separations were carried out with chloroform~metl~anol (95:5) using glass fiber paper impregnated with silicic acid (Chromar 500, Rlallinckrodt Chemical Co.). This solvent system was useful for preliminary screening of plasma extracts. The solvent systems recommended by Sunshine’ for urine are equally useful for analysis of plasma by TLC. The remainder of the methanol solution was used for analysis by GC and GC-MS procedures. DericatiTle fonnatio?z In order to carry out GC analysis it is best to convert drugs, drug metabolites and other compounds present in the methanol solution to suitable derivatives. Methylation followed by silylation usually provides derivatives with good gas chromatographic properties.
ISOLATION
OF DRUG METABOLITES
FROM PLrlSMA
383
M~thVllZtiO~Z i
An ethereal solution of diazomethane (prepared by ether codistillation from Diazald, Aldrich Chemical Co.) was added to the methanol solution (0.4-0.5 ml) until the yellow color persisted. After standing at room temperature for 15 min, the excess ether, diazomethane and methanol were removed with a nitrogen stream. The residue was dissolved in 30 ~1 of pyridine. Silylation
his-Trin~etl~ylsilylacetamide (BSA) (30~1) or b is -t rimethylsilyltrifluoroacetamide (BSTFA) (30 ~1) and IO ,~l of trimethylchlorosilane (TMCS) were added to the pyridine solution of methylated drugs and drug metabolites. After heating at 60” for 1-2 h, an aliquot (2-j ~1) was used directly for analysis. Gas chromatogra$hy Gas chromatographic analyses were carried out with Barber-Colman model 5000 instruments equipped with Keithley model 417 picoammeters and Texas Instruments recorders. The columns were 12 ft x 4 mm glass \V columns. The column packing was 5% SE-30 on 80-100mesh Gas Chrom P prepared according to the usual procedure of this laboratory”. The injector temperature was 260”; the detector bath temperature was 300~. Typical gas pressures were nitrogen, 26 psi; air, 40 psi; hydrogen, 15 psi. The nitrogen flow rate was 40-50 ml per min at 200~. The separations were carried out by temperature programming from 90’ to 250 ’ at z”/min.
Mass spectra were obtained with an LKB model gooo gas chromatograph~mass spectrometer. The column was a 9 ft x 4 mm glass coil with a I yO SE-30 column packing. The ionizing current was 60 PA; the voltage was 70 eV; the ion source was at 270’; the usual scan time was 3 to 6 sec. Removal
of fatty
acids
The methanol extract (0.4-0.5 ml) was diluted to I ml with methanol and extracted with 5 ml of iso-octane; a r5-ml screw-capped (Teflon-lined) centrifuge tube was used for the extraction. After mixing vigorously the contents of the tube were centrifuged for 5 min, and then chilled for 5 min in an ice bath. The clear supernatant (iso-octane) was removed with a Pasteur pipette and the extraction procedure was repeated with an additional 5 ml of iso-octane. The combined iso-octane extract may be analyzed for fatty acids if desired. The purpose of this step is to decrease the amount of fatty acids in the sample without altering the amounts of drugs and drug metabolites which may be present.
The recovery of drugs and drug metabolites from plasma was followed by injecting radioactive drug diluted with carrier compound intraperitoneally into a rat and withdrawing blood after 30 min. Twenty-two mg of phenobarbital (sodium salt) was dissolved in 0.5 ml of distilled water and added to a solution of 2 pC of radioactive phenobarbital ([z-X]phenobarbital dissolved in 0.1 ml of ethanol). The solution of radioactive and carrier phenobarbital was injected intraperitoneally into a 200-g male rat. After 30 min, the rat was anesthetized with an intraperitoneal injection of 0.25 ml (12.5 mg) of pentobarbital Clilz. Chim.
Acta,
37 (1972)
381-3X6
flOl
384
c‘f (ai.
(sodium salt). After approximately 5 min, blood (5 --6 ml) was withdrawn by cardiac puncture into a lleparinized syringe and centrifuged imnlediatel~. Twenty mg of caffeine was dissoIved in 0.5 ml of distilled water and added to z $I of rr-14C-methvl-caffeine dissolved in 0.1 ml of distilled water. The solution of radioactive and carrier caffeine was injected intraperitoneally into a zoo-g male rat. The plasma was obtained from the rat f(~llo~vin~ the procedure for ~~~~ex~o~~al-bital. The radioactive plasma obtained from the plrenobarbitaland caffeine-trcatctl rats was extracted with three 1-1711portions of isopropanol as described. The recover) of both phenobarbital and caffeine (and metabolites) as measured b!r radioactivit!~ comparisons RESUI,TS
ANil>
in the isopropanol
extracts
was xoo
:- 5”; ~du~~licate esperil~lents).
nISCUSSlON
A gas chrolnato~rapll~c analysis of an isoprnpanol extract of I ml of plasma obtained from a rat treated with phenobarbital and !z-l*C]phenobarbital ii shown in Fig. I. Since the rat had been anesthetized with pentobarbital, both pentobarbital and phenobarbital were present in the gas chromatographic tracing. The structures of the two barbiturates were confirmed by CC-3%. The other major peaks in Fig. I wvero identified by CC-MS as the methyl esters of palmitic (C : 16 : o), oleic (C : IS : I), linoleic: (C:I~:Z) and stearic (C:IS:O) acids. A gas cllrolllato~raphi~ analysis of an isopropanol extract of T 1x1 of plasma obtained from a rat treated with caffeine and jx-lICjmetllg-l-caffeirlc and anc~sthetizcrl with pentobarbital is shown in Fig. 2. Caffeine, pentobarbital, palmitic, oleic, linolcic and stearic acids were identified by CC--MS. The recovery of both ~)llenobarbital and caffeine, and their metaholitrs, from the plasma was followed by comparing the amount of radioactivit>r present in the combined isopropanol extracts with the total radioactivity of the plasma. Three 1-1171 extractions with isopropanol were found to be sufficient for the cluantitative removal of caffeine, ~llenob~lrbital, and their illetabolites from the diluted plasma. The rccover-y of radioactivity in the combined isopropanol extracts was roe -i_ ~$0 in each. Fatt!. acid methyl esters do not interfere with the quantiiication or identification of caffeine, pentobarbital or phenobarbital. Howevtr, drugs and drug rnctabolites with rnet~~~lene unit (&I.L7.) values close to those of the usual fatt\T acid rnetlrvl esters lK$03-EXT)
RAT PLASMA-PHENOBARBITAL ME 5%
TMS
SE-30
TP2’1MIN
(SO”1
C 1’8 I
PHENO PENT0
TIME-MIN
Fig. I. Gas chromatographic analysis 01 the isopropanul extract ul’ I 1111of plasms from a rat treated with phenob~~rbital and anesthetized with pentol~arbi~~l. The con~pound~ were separatcti
as the MMe-TMSi derivatives by temperature programmin g from go’ at L”jmin on c212 ft 5”(, SE-Jo column. The peaks identified by GC-MS are PENTO, pentoharbital; PHENO, phenobarbital; C: 16:0, palmitic acid; C: 18:r, oleic acid: C: 18:3, linolcic acid and C: IS:O, stearic ncld.
ISOLATION
OF DRUG METABOLITES
would be difficult with hexane
to identify.
or iso-octane,
The extraction
FROM PLASMA
Fatty
3%
acids can be extracted
from methanol
solution
and this step was included for the analysis shown in Fig. 3.
of fatty
acids from the methanol
Tolution
by iso-octane
was
followed by adding [I-Xlpalmitic acid to a methanol extract of human plasma containing phenobarbital. Two extractions with iso-octane removed g5”/, of the radioactive palmitic acid. m’hen radioactive phenobarbital was added to a similar methanol solution,
instead
of [r-l%Zjpalmitic
acid, only 4%
of the radioactive
phenobarbital
was present in the first iso-octane extract and 37 in the second extract. Thus, about 059s of the fatty acids can be removed by iso-octane extraction, and goPg5y0 of phenobarbital which is present will remain in the methanol solution. Fig. 3 shows a GC analg.sis of (I) a methanol extract of human plasma which contained phenobarbital and phenobarbital metabolites, and (2) an iso-octane extract of the methanol solution. Both analyses were carried out after conversion of all components to methyl (Me) and methyl-trimethylsilyl (MeeTMSi) derivatives. The charts are superimposed to provide a direct comparision. The iso-actane solution shows only a trace content of phenobarbital. The fatty acids were extracted along with smaller amounts of unidentified substances. The fact that the iso-octane solutions as well as the methanol solutions can be analyzed by GC techniques makes it possible in each instance
to determine
if there is a loss of drugs or drug metabolites
when this step is
employed. Experience with this procedure, with and without the additional step of isooctane extraction, has shown that the initial extraction step with isopropanol is highly effective for the isolation of neutral and basic drugs and drug metabolites from plasma. Some acidic substances are apparently displaced from protein-bound complexes and may also be extracted; fatty acids, for example, are present in the extract. The procedure is rapid and simple, and no failures in extraction due to emulsion formation have been encountered. It is possible to apply separation, identification and quantification methods directly or after derivative formation. \Ve prefer gas phase analytical
RAT
c
PLASMA-CAFFEINE ME-TM3
5%
SE-X)
(K2C03-
CnFF TP 29MIN
I8 I
EXTI
60
70 TIME-MIN
II c
16 0
(75)
(TMSI C.16 0
50
P
60
180
l;ig. 2. (Gas chromatographic analysis of the isopropanol cxtract of I ml of plasma from a rat treated with cafficine and anesthetized with pentobarbital. The peaks identified by GC-MS are PENTO, pentobarbital; CAFF, caffeine; C: 16:0, palmitic acid; C: 18: I, olcic acid; C: 18 :z, linolcic acid and C : IX :o, stearic acid. The conditions for separation by temperature programming from 7.j0 were the same as those described in Fig. I.
HUMAN
PLASMA-
PHENOBARBITAL
(K&O,
EXT)
ME-TMSI 5% SE-30
TP Z”/MIN
MEOH ISO-OCT---
70
80
90
TIME-MIN
Fig. 3. Analysis of human plasma after phenobarbital mgcstion. The x gas chromatographic analysi\ of the methanol solution of drugs and drug metabolitcs extracted l’rom pla.;ma by isopropanol IS represented by the solid hnc; the analysis of the first ix-octane estract of the methanol solution of drugs and drug metabolites is reprcsentctl by the broken line. The contlitions tor separation were the same as those described for Fig. I. The peaks Identified by GC-MS wcrc PHEXO, phenobarbital; C: 16:o, palmitic acid; C: 18: I, oleic acid; c’ : 18: 2, linoleic acid and C: r8:0, stearlc acid. Very little phenobarbital (PHENC)) was extracted into the iso-octane phase lrom methanol.
procedures
after derivative
formation,
chiefly
because
this is the preferred
method
when drug metabolites are under study. Lipid-soluble materials (primarily fatty acids) may be removed by iso-octane extraction of a methanol solution of the extracted materials. This step may or may not be necessarv. It is not possible to reverse the order of extractions; if k-octane is used for the ktial extraction of a plasma samplt an emulsion with undesirable properties may be formed. Other alcohols may be used in the plasma extraction process; ethanol is satisfactory, according to SunshineI, and it is possible that propanol or butanol could be used. The isopropanol-potassium carbonate combination seems to be particularly satisfactory in combining effecti\,{ extraction with effectilre separation of phases, and retention of protein at the watclr interface. lt is also possible to apply this method to urine samples: procedure’ was developed for urinary analysis.
This work was supported General Medical Sciences.
by grant
G&I-16216
the original Sunshin~~
of the National
Institute
of
CCA
CHIMICA
381
.4CTA
4714
A RAPID
PROCEDURE
~IET~~3OLIT~S
FROM
FOR
THE
ISOLATION
01;
DRUGS
AND
DRUG
PLASMA
SUMMARl
A rapid procedure for the extraction of neutral and basic drugs and drug metabolites with isopropanol from diluted plasma saturated with potassium carbonate has been developed. The isopropanol extract is suitable for analysis by thin layer or gas chromatography. Recovery experiments (radioactivity) with phenobarbital and caffeine
gave
IOO +
5%
recovery.
The absence
of emulsions
contributes
to the high
recovery.
INTRODUCTION
Many procedures for isolating neutral and basic drugs and drug metabolites from plasma or serum have been published. Almost invariably, the problem of emulsion formation is ignored or centrifugation is recommended. When normal plasma or serum is analysed, centrifugation will usually keep emulsion formation within acceptable limits. In clinical studies, however, the chemist is concerned more often than not with abnormal plasma, and intractable emulsions which do not respond to centrifugation or other laboratory manipulations are frequently obtained. Reliable quantitative data are unobtainable under these circumstances. This di~culty has been resolved by applying to plasma the salting-out procedure developed by Sunshine and co-workers1 for the rapid isolation of drugs and drug metabolites from urine. Diluted plasma, to which a small volume of isopropanol has been added, is saturated with potassium carbonate. IJpon centrifugation, the isopropanol layer containing drugsand drug metabolites separates as the upper phase. Proteins precipitated by the isopropanol and potassium carbonate are found at the interface. The isopropanol extract can be analyzed directly, or after transfer to methanol solution, by thin layer chromatography (TLC) ; it is possible to carry out preliminary screening for drug metabolites on large numbers of samples by TLC if this is desired. After conversion to suitable derivatives, compounds in the isopropanol fraction can Clin. Chirn. Acta, 37 (1972j
381-3X6
be analyzed by gas chromatography (GC), and gas chronlatograpll~~nl~~~s spectrometry (GCMS). A gas chromatograph-mass spectrometerPcomputer instrument (GC MSPCOM) is particularly useful for tllis work. Gas chrornatograpllic analysis provides a quantitative analysis as well as tentative identification of the drugs and drug metabolites present in plasma. X11 structural assignments, howcvcr, shnuld be con firmed by mass spectromctric
analysis.
EXPERIMENTAL
Heparinized
rat blood was obtained
by cardiac puncture
with pentobarbital. The plasma was separated immediately stored at - 13” until analyzed. Isopropanol, methanol and iso-octane were Nanogradc Chemical Co.). Anhydrous
potassium
carbonate
(granular)
was obtained
from rats anesthetized by- centrifugation quality
and
(Mallinckrodt
from Fisher Chemical
co. Isolatiol2 f~fom jdasma One ml of plasma was transferred to a x5-ml screw-capped centrifuge tube and diluted with 5 ml of glass-distilled water. One ml of isopropanol \vas added, followed by 6 g of anhydrous granular potassium carbonate. The contents of the tube were rapidly and thoroughly mixed by inversion; the solution was agitated in a Vortex mixer for zo set and then allowed to stand for 5 min. The phases separated during this time and the separation was completed by centrifugation for 5 min. The isopropanol layer (upper phase, 0.9 ml) was transferred wit11 a Pasteur pipette to a small scrcwcapped (Teflon-lined) tube (12 mm r: xoo mm). Tlie extraction was repeated twice by adding I ml of isopropanol to the potassium carbonate solution, mixing thoroughly with a \Tortex mixer and centrifuging for 5 min. The combined isopropanol extracts (approximately 3 ml) containing drugs and drug metabolites was evaporated ivith the aid of a nitrogen stream. The residue was dissolved in 0.5 ml of methanol for subsequent analysis by TLC or GC methods. Thin
layer chromatograf$ An aliquot (50-100 ~1) of the methanol solution (0.5 ml) was used for analysis by TLC. The separations were carried out with chloroform~metl~anol (95:5) using glass fiber paper impregnated with silicic acid (Chromar 500, Rlallinckrodt Chemical Co.). This solvent system was useful for preliminary screening of plasma extracts. The solvent systems recommended by Sunshine’ for urine are equally useful for analysis of plasma by TLC. The remainder of the methanol solution was used for analysis by GC and GC-MS procedures. DericatiTle fonnatio?z In order to carry out GC analysis it is best to convert drugs, drug metabolites and other compounds present in the methanol solution to suitable derivatives. Methylation followed by silylation usually provides derivatives with good gas chromatographic properties.
ISOLATION
OF DRUG METABOLITES
FROM PLrlSMA
383
M~thVllZtiO~Z i
An ethereal solution of diazomethane (prepared by ether codistillation from Diazald, Aldrich Chemical Co.) was added to the methanol solution (0.4-0.5 ml) until the yellow color persisted. After standing at room temperature for 15 min, the excess ether, diazomethane and methanol were removed with a nitrogen stream. The residue was dissolved in 30 ~1 of pyridine. Silylation
his-Trin~etl~ylsilylacetamide (BSA) (30~1) or b is -t rimethylsilyltrifluoroacetamide (BSTFA) (30 ~1) and IO ,~l of trimethylchlorosilane (TMCS) were added to the pyridine solution of methylated drugs and drug metabolites. After heating at 60” for 1-2 h, an aliquot (2-j ~1) was used directly for analysis. Gas chromatogra$hy Gas chromatographic analyses were carried out with Barber-Colman model 5000 instruments equipped with Keithley model 417 picoammeters and Texas Instruments recorders. The columns were 12 ft x 4 mm glass \V columns. The column packing was 5% SE-30 on 80-100mesh Gas Chrom P prepared according to the usual procedure of this laboratory”. The injector temperature was 260”; the detector bath temperature was 300~. Typical gas pressures were nitrogen, 26 psi; air, 40 psi; hydrogen, 15 psi. The nitrogen flow rate was 40-50 ml per min at 200~. The separations were carried out by temperature programming from 90’ to 250 ’ at z”/min.
Mass spectra were obtained with an LKB model gooo gas chromatograph~mass spectrometer. The column was a 9 ft x 4 mm glass coil with a I yO SE-30 column packing. The ionizing current was 60 PA; the voltage was 70 eV; the ion source was at 270’; the usual scan time was 3 to 6 sec. Removal
of fatty
acids
The methanol extract (0.4-0.5 ml) was diluted to I ml with methanol and extracted with 5 ml of iso-octane; a r5-ml screw-capped (Teflon-lined) centrifuge tube was used for the extraction. After mixing vigorously the contents of the tube were centrifuged for 5 min, and then chilled for 5 min in an ice bath. The clear supernatant (iso-octane) was removed with a Pasteur pipette and the extraction procedure was repeated with an additional 5 ml of iso-octane. The combined iso-octane extract may be analyzed for fatty acids if desired. The purpose of this step is to decrease the amount of fatty acids in the sample without altering the amounts of drugs and drug metabolites which may be present.
The recovery of drugs and drug metabolites from plasma was followed by injecting radioactive drug diluted with carrier compound intraperitoneally into a rat and withdrawing blood after 30 min. Twenty-two mg of phenobarbital (sodium salt) was dissolved in 0.5 ml of distilled water and added to a solution of 2 pC of radioactive phenobarbital ([z-X]phenobarbital dissolved in 0.1 ml of ethanol). The solution of radioactive and carrier phenobarbital was injected intraperitoneally into a 200-g male rat. After 30 min, the rat was anesthetized with an intraperitoneal injection of 0.25 ml (12.5 mg) of pentobarbital Clilz. Chim.
Acta,
37 (1972)
381-3X6
flOl
384
c‘f (ai.
(sodium salt). After approximately 5 min, blood (5 --6 ml) was withdrawn by cardiac puncture into a lleparinized syringe and centrifuged imnlediatel~. Twenty mg of caffeine was dissoIved in 0.5 ml of distilled water and added to z $I of rr-14C-methvl-caffeine dissolved in 0.1 ml of distilled water. The solution of radioactive and carrier caffeine was injected intraperitoneally into a zoo-g male rat. The plasma was obtained from the rat f(~llo~vin~ the procedure for ~~~~ex~o~~al-bital. The radioactive plasma obtained from the plrenobarbitaland caffeine-trcatctl rats was extracted with three 1-1711portions of isopropanol as described. The recover) of both phenobarbital and caffeine (and metabolites) as measured b!r radioactivit!~ comparisons RESUI,TS
ANil>
in the isopropanol
extracts
was xoo
:- 5”; ~du~~licate esperil~lents).
nISCUSSlON
A gas chrolnato~rapll~c analysis of an isoprnpanol extract of I ml of plasma obtained from a rat treated with phenobarbital and !z-l*C]phenobarbital ii shown in Fig. I. Since the rat had been anesthetized with pentobarbital, both pentobarbital and phenobarbital were present in the gas chromatographic tracing. The structures of the two barbiturates were confirmed by CC-3%. The other major peaks in Fig. I wvero identified by CC-MS as the methyl esters of palmitic (C : 16 : o), oleic (C : IS : I), linoleic: (C:I~:Z) and stearic (C:IS:O) acids. A gas cllrolllato~raphi~ analysis of an isopropanol extract of T 1x1 of plasma obtained from a rat treated with caffeine and jx-lICjmetllg-l-caffeirlc and anc~sthetizcrl with pentobarbital is shown in Fig. 2. Caffeine, pentobarbital, palmitic, oleic, linolcic and stearic acids were identified by CC--MS. The recovery of both ~)llenobarbital and caffeine, and their metaholitrs, from the plasma was followed by comparing the amount of radioactivit>r present in the combined isopropanol extracts with the total radioactivity of the plasma. Three 1-1171 extractions with isopropanol were found to be sufficient for the cluantitative removal of caffeine, ~llenob~lrbital, and their illetabolites from the diluted plasma. The rccover-y of radioactivity in the combined isopropanol extracts was roe -i_ ~$0 in each. Fatt!. acid methyl esters do not interfere with the quantiiication or identification of caffeine, pentobarbital or phenobarbital. Howevtr, drugs and drug rnctabolites with rnet~~~lene unit (&I.L7.) values close to those of the usual fatt\T acid rnetlrvl esters lK$03-EXT)
RAT PLASMA-PHENOBARBITAL ME 5%
TMS
SE-30
TP2’1MIN
(SO”1
C 1’8 I
PHENO PENT0
TIME-MIN
Fig. I. Gas chromatographic analysis 01 the isopropanul extract ul’ I 1111of plasms from a rat treated with phenob~~rbital and anesthetized with pentol~arbi~~l. The con~pound~ were separatcti
as the MMe-TMSi derivatives by temperature programmin g from go’ at L”jmin on c212 ft 5”(, SE-Jo column. The peaks identified by GC-MS are PENTO, pentoharbital; PHENO, phenobarbital; C: 16:0, palmitic acid; C: 18:r, oleic acid: C: 18:3, linolcic acid and C: IS:O, stearic ncld.
ISOLATION
OF DRUG METABOLITES
would be difficult with hexane
to identify.
or iso-octane,
The extraction
FROM PLASMA
Fatty
3%
acids can be extracted
from methanol
solution
and this step was included for the analysis shown in Fig. 3.
of fatty
acids from the methanol
Tolution
by iso-octane
was
followed by adding [I-Xlpalmitic acid to a methanol extract of human plasma containing phenobarbital. Two extractions with iso-octane removed g5”/, of the radioactive palmitic acid. m’hen radioactive phenobarbital was added to a similar methanol solution,
instead
of [r-l%Zjpalmitic
acid, only 4%
of the radioactive
phenobarbital
was present in the first iso-octane extract and 37 in the second extract. Thus, about 059s of the fatty acids can be removed by iso-octane extraction, and goPg5y0 of phenobarbital which is present will remain in the methanol solution. Fig. 3 shows a GC analg.sis of (I) a methanol extract of human plasma which contained phenobarbital and phenobarbital metabolites, and (2) an iso-octane extract of the methanol solution. Both analyses were carried out after conversion of all components to methyl (Me) and methyl-trimethylsilyl (MeeTMSi) derivatives. The charts are superimposed to provide a direct comparision. The iso-actane solution shows only a trace content of phenobarbital. The fatty acids were extracted along with smaller amounts of unidentified substances. The fact that the iso-octane solutions as well as the methanol solutions can be analyzed by GC techniques makes it possible in each instance
to determine
if there is a loss of drugs or drug metabolites
when this step is
employed. Experience with this procedure, with and without the additional step of isooctane extraction, has shown that the initial extraction step with isopropanol is highly effective for the isolation of neutral and basic drugs and drug metabolites from plasma. Some acidic substances are apparently displaced from protein-bound complexes and may also be extracted; fatty acids, for example, are present in the extract. The procedure is rapid and simple, and no failures in extraction due to emulsion formation have been encountered. It is possible to apply separation, identification and quantification methods directly or after derivative formation. \Ve prefer gas phase analytical
RAT
c
PLASMA-CAFFEINE ME-TM3
5%
SE-X)
(K2C03-
CnFF TP 29MIN
I8 I
EXTI
60
70 TIME-MIN
II c
16 0
(75)
(TMSI C.16 0
50
P
60
180
l;ig. 2. (Gas chromatographic analysis of the isopropanol cxtract of I ml of plasma from a rat treated with cafficine and anesthetized with pentobarbital. The peaks identified by GC-MS are PENTO, pentobarbital; CAFF, caffeine; C: 16:0, palmitic acid; C: 18: I, olcic acid; C: 18 :z, linolcic acid and C : IX :o, stearic acid. The conditions for separation by temperature programming from 7.j0 were the same as those described in Fig. I.
HUMAN
PLASMA-
PHENOBARBITAL
(K&O,
EXT)
ME-TMSI 5% SE-30
TP Z”/MIN
MEOH ISO-OCT---
70
80
90
TIME-MIN
Fig. 3. Analysis of human plasma after phenobarbital mgcstion. The x gas chromatographic analysi\ of the methanol solution of drugs and drug metabolitcs extracted l’rom pla.;ma by isopropanol IS represented by the solid hnc; the analysis of the first ix-octane estract of the methanol solution of drugs and drug metabolites is reprcsentctl by the broken line. The contlitions tor separation were the same as those described for Fig. I. The peaks Identified by GC-MS wcrc PHEXO, phenobarbital; C: 16:o, palmitic acid; C: 18: I, oleic acid; c’ : 18: 2, linoleic acid and C: r8:0, stearlc acid. Very little phenobarbital (PHENC)) was extracted into the iso-octane phase lrom methanol.
procedures
after derivative
formation,
chiefly
because
this is the preferred
method
when drug metabolites are under study. Lipid-soluble materials (primarily fatty acids) may be removed by iso-octane extraction of a methanol solution of the extracted materials. This step may or may not be necessarv. It is not possible to reverse the order of extractions; if k-octane is used for the ktial extraction of a plasma samplt an emulsion with undesirable properties may be formed. Other alcohols may be used in the plasma extraction process; ethanol is satisfactory, according to SunshineI, and it is possible that propanol or butanol could be used. The isopropanol-potassium carbonate combination seems to be particularly satisfactory in combining effecti\,{ extraction with effectilre separation of phases, and retention of protein at the watclr interface. lt is also possible to apply this method to urine samples: procedure’ was developed for urinary analysis.
This work was supported General Medical Sciences.
by grant
G&I-16216
the original Sunshin~~
of the National
Institute
of