Determination medazepam (Nobrium), diazepam (Valium), and their

Determination of Medazepam (Nobrium), Diazepam (Valium) and Th Biotransformation Product and Urine by Electron Capture Gas-Liquid Chromatography J. Arthur F. de Silva and Carl V. Puglisi Department of Clinical Pharmacology, Hoffman-La Roche Inc., Nutley, N. J . 07110

A sensitive and specific electron capture gas chromatographic assay was developed for the determination of medazepam and its major metabolites in blood and urine. It involves the selective extraction of the intact compounds into diethyl ether from blood buffered to pH 9.0 and from urine (following incubation at 37 "C for 2 hours at pH 5.3 with glucuronidase), made alkaline with NaOH. The assay has an overall recovery of medazepam of 94.5% & 5.2 from blood and 80.0% =k 4.5 from urine. The recovery of the major metabolites present in blood, diazepam and N-desmethyl diazepam is 86"0% & 5.8 and 94.0% + 6.0, respectively. The recovery of the major urinary metabolites oxazepam, Ndesmethyl diazepam and 3-hydroxy diazepam when added to urine as authentic reference standards ranged from 80-85%. The sensitivity limits of detection are of the order of 40-50 ng medazepam/ml of blood or urine. These limits can be increased almost 5-fold by using a 4- or 5-ml specimen per assay if needed. The method was applied to the determination of blood levels and the urinary excretion pattern in man following single and multiple 50-mg oral doses of Nobrium.

MEDAZEPAM (the active drug substance in Nobrium marketed by F. Hoffman-La Roche and Co. A.G. Basle, Switzerland) 7-chloro-2,3-dihydro-l-rnethyl-5 -phenyl- 1H-1,4- benzodiazepine hydrochloride ( I , 2 ) is a member of the 1,4-benzodiazepine series of compounds and is analogous to chlordiazepoxide, and diazepam. (Chlordiazepoxide and diazepam are the active drug substances in Librium and Valium, respectively, marketed by Hoffmann-La Roche Inc., Nutley, N. J., U.S.A.) It differs from diazepam in having a methylene (-CHz-) group at position 2 instead of a carbonyl group, Figure 1. The pharmacological properties of the compound have been reported (3, 4) and it is currently marketed as an anti-anxiety agent. Studies by Schwartz and Carbone (5) on the identification of the biotransformation products present in human, rat, and dog blood and urine following the oral administration of 14C-labelled medazepam (6) showed that the compound was rapidly and extensively metabolized producing measurable amounts of several metabolites in the blood and urine, Figure 1. Consequently, a specific and sensitive method was required which could differentiate medazepam from these metabolites and measure them quantitatively. (1) L. H. Sternbach, E. Reeder, and G. A. Archer, J. Org. Cjzem., 28, 2456 (1963). (2) G. A. Archer and L. H. Sternbach, Chem Reu., 68,747 (1968). (3) L. 0. Randall, W. Schallek, C. Scheckel, R. Banziger, andR. A. Moe, Arzneim. Forsch., 18, 1542 (1968). (4) L. 0. Randall, C. Scheckel, and W. Pool, Arch. Int. Pharmcodyn., 185, 135 (1970). (5) M. A. Schwartz and J. J. Carbone, Biochem. Pharmacol., 19, 343 (1970). (6) H. H. Kaegi, J. Label. Compounds, 3, 493 (1967).

During the investigation of suitable analytical parameters fortheanalysis of medazepam, its response to electron capture/ gas-liquid chromatography (ECIGLC) was found to be sufficiently sensitive for quantitation in the nanogram gram) range of sensitivity. Although medazepam and diazepam are structurally analogous compounds, the presence of the methylene (-CH2-) group in position 2 in the benzodiazepine ring stabilizes the compound to acid hydrolysis (6N HCl) whereas diazepam is quantitatively converted to 2 methyl amino-5-chlorobenzophenone (MACB). Because of the presence of several metabolites which could be hydrolyzed to yield the same respective end products MACB and ACB (Figure l), the published procedure for diazepam and its N-desmethyl metabolite (7, 8) was not suitable for the determination of medazepam and its metabolites because of lack of specificity. Furthermore, the temperature limitations on the use of electron capture detectors containing Titanium Tritide (3H) as the radioactive source, and of Carbowax 20M-TPA, used as the liquid phase in the column, reduced the sensitivity and usefulness of the assay for the determination of medazepam. The use of high temperature stable liquid phases such as the phenyl silicones (OV-17 and OV-25) and the 03Ni electron capture detector enabled the quantitation of the intact benzodiazepines and their -2-ones with the desired sensitivity and specificity required for therapeutic concentrations of these compounds. The GLC analysis of intact benzodiazepines was reported by Marcucci et al. (9) using a flame ionization detector which per se was not suitable for the quantitation of blood levels following therapeutic doses. The specific and sensitive GLC assay reported here uses a more polar liquid phase (OV-17) which completely resolves all the benzodiazepine compounds present and thus enables the specific and highly sensitive quantitation of these compounds utilizing the 63Nielectron capture detector in the pulsed dc operational mode. The method was applied to the determination of blood level curves and the urinary excretion in man following single and multiple 50-mg oral doses of Nobrium. EXPERIMENTAL

Analysis for Medazepam and Its Major Metabolites in Blood and Urine. Parameters for GLC Analysis. COLUMN.The column packing was a preconditioned phase containing 3% OV-17 on 60/80 mesh Gas Chrom Q (Applied Science Labs, (7) J. A. F. de Silva, M. A. Schwartz, V. Stefanovic, J. Kaplan, and L. D'Arconte, ANAL.CHEM., 36, 2099 (1964). (8) J. A. F. de Silva, B. A. Koechlin, and G. Bader, J . Phnrm. Sci., 55, 692 (1966). (9) F. Marcucci, R. Fanelli, and E. Musini J. Chromatogr., 37,318 (1968).

ANALYTICAL CHEMISTRY, VOL. 42, NO. 14, DECEMBER 1970

1725

CI

N-DESMETHYL MEDAZEPAM

II

aNH

do

N-DESMETHYL-I.2DEHYORO-MEDAZEPAM 'JII

ACE

I

Figure 1. Chemical reactions and metabolic pathways of medazepam in man (M) and in the dog (D)

CI

hHZ \

I c=o

CJ DIAZEPAM

MAC0

a

III

N-DESMETHYL

DIAZEPAM

P

ACHB

IX

F"!,

X

N

CI

- C-0

N -C=O

1

>WOH

$ =N

3 - HYDROXY

- DIkZEPAM

State College, Pa.) packed in a U-shaped %foot, 4-mm i.d. borosilicate glass column. The column was further conditioned at 325 "C for 4 hours with "no flow" of carrier gas, followed by 12 hours at 215 "C with carrier flowing at 40 ml/min. The useful life span of such a column was about 4-5 months of continuous use. INSTRUMENTAL PARAMETERS. A Micro-Tek Gas Chromatograph, Model MT-220 equipped with a GaNielectron capture

=N

CI

XI

OXAZEPAM 375

detector containing a 10 mCi G8Ni p ionization source was used, Argon-Methane (90 :10) Matheson, (oil pumped and dry) was used as the carrier gas, the column head pressure being adjusted to 40 psig and the flow rate to 100-110 ml/min with the detector purge gas adjusted to 20 ml/min. The temperature settings were as follows: Oven 230 "C, injection port 270 OC, detector 310 "C. The conditions of column head pressure, flow rate, and oven temperature must be adP

n

I

I

I Medorepom II n-desmethyl Medazepam IY DiorePom

Figure 2. Gas chromatograms of diethyl ether extracts of ( A ) patient control blood; ( B ) control blood containing added authentic standards, and (C) patient blood post medication.

RETENTION ?lME E R t )

1726

0


I N MINUTES

Figure 3. Calibration curves of medazepam and its major metabolites

5

I

0 7 5 I.25 10 2.0 10

15 0 2 50

22 5 3 75

20

30

4 0 2 0

6 0

I,2-dehydro

Medateporn

-I

30

CONCENTRATION I N N A N O G R A M S

Table I. Chemical Names and Physical Properties of Compounds Referred to Compound I I1 I11

IV V

VI VI1

Generic name Medazepam N-Desmethyl medazepam N-Desmethyl-l,2dehydro-medazepam Diazepam N-Desmethyldiazepam 3-Hydroxydiazepam Oxazepam

VI11

-

IX

-

X

-

Chemical name 7-chloro-2,3-dihydro-l-methyl-5phenyl-1H-I ,4-benzodiazepine 7-chloro-2,3-dihydro-5-phenyl1H-1,4benzodiazepine 7-chloro-5-phenyl-3H-l,4-benzodiazepine 7-chloro-l,3-dihydro-I-methyl-5-

phenyl-2H-1,4-benzodiazepin-2-one 7-chloro-1,3-dihydro-5-phenyl-2H-1,4benzodiazepin-Zone 7-chloro-l,3-dihydro-3-hydroxy-I-methyl5-phenyl-2H-l,4-benzodiazepin-2-one 7-chloro-l,3-dihydro-3-hydroxy-5-phenyl-

2N-1,4-benzodiazepin-Zone 2-amino-5-chlorobenzoahenone (ACB) 2-amino-3-hydroxy-5-chlorobenzophenone (ACHB) 2-methylamino-5-chlorobenzophenone (MACB)

justed so as to obtain a retention time ( R J of 4.2 minutes for medazepam. Under these conditions the retention times of diazepam and N-desmethyl diazepam are 8.7 and 12.6 minutes, respectively. A typical chromatogram is shown in Figure 2. The solid state electrometer (Model No. 8169) input was set at IO2and the output attentuation was 32 giving a response of 3.2 x 10-9 A for full scale deflection (fsd), the chart speed was 30 in./hour, and the time constant on the 1.0 mV Honeywell recorder (Model No. 194) was 1 second (fsd). The response of the 5*Ni EC detector (operated in the pulsed dc mode) to medazepam and diazepam showed maximum sensitivity at 45 volts dc at a 270-psec pulse rate and a 4-psec pulse width. Under these conditions, 20 ng of medazepam and 4 ng of diazepam give nearly full-scale pen response on the 1.0-mV recorder. The minimum detectable amount of medazepam is 0.04 to 0.05 pg/ml of blood or urine. CALIBRATION OF MEDAZEPAM AND DIAZEPAM by GLC. A calibration (external standard) curve of the peak areas of

Mp T 95-47 256.72

170-171

254.73

i 01- 104

284.74

131-135

270 72

21 6-217

300 15

I IS-l20

286.72

265-206

211.67

97-98

247,35

163-167

245 72

94-95

I

medazepam or diazepam us. c per 1 0 p l o f 2 0 ~ a c e t o t t e ' as shown in Figure 3. spective benzodiazepine mined using the formula, I / I , (change in standing currefit) = or Peak Area [height (cm) X

Assay in Blood, PREPARATI~N dk S + A N ~ ) A%Rt U~T r U ~ s . The respective benzodiazepines mid bcrtzodiazepin-2-ones


6

1787

solution. This solution is 1Mwith respect to H&3O3-Na2CO3 KCl. This solution should be stored at about 35-37 "C to prevent crystallization of the salts out of the solution. Diethyl ether, analytical reagent grade ether (absolute) containing not more than 0.0005 % residue after evaporation and peroxide content not more than 0.0005 (Malliackrodt) ng lacng lacmust be used from a can opened no more than 5 days premedmedviously. azepam __ Scintillometry azepam % The following were also used : hexane-Pesticide grade, added1 DPM DPM rererecovered recovery covered covery Fisher "W-300" certified hexanes; acetone, reagent grade, ml added stored over anhydrous sodium sulfate; ethanol, Absolute 98 88 88 4,064 100 4,136 ethanol; and 2.0 and 6.0N WCI; and 1.0, 2.0, and 10.ON 194 97 7,903 102 200 7,778 NaOH were made up with distilled water. 95 300 100 12,468 11,872 300 370 93 94 14,508 400 15,414 Procedure. Into a 40-ml stoppered centrifuge tube, add Meanrecovery 9 7 . 3 % =k 3 . 6 9 4 . 5 % =t5 . 2 1.0 mi of blood, 5 ml of pW 9.0 borate buffer, and 10 ml of diethyl ether for the first extraction. Seal the stopper with B. Recovery of Diazepam and N-Desmethyl Diazepam distilled water, and extract by shaking for 10 minutes on a Determined by Gas Chromatography Using the Relative reciprocating shaker. Centrifuge the samples for 10 minutes. Calibration Technique Repeat the extraction procedure with another 10-rnl portion Reof ether, centrifuge, and combine the ether extracts. Along Added, Recovered, covery, with the samples run a specimen of control blood (taken prefCompound ng ng % erably from the patient prior to medication) and duplicate Diazepam 10 9.0 90 1-ml specimens of control blood containing 200 ng of med20 15 8 79 azepam, 300 ng of N-desmethyl medazepam, 40 ng of diaze30 24 6 82 pam, and 80 ng of N-desmethyl diazepam (0.1 ml of solution 30 27.4 91 D) added as internal standards. Add 5.0 ml of 2.ON HCl to 40 36.8 92 the combined ether extracts, shake for 10 minutes, and cen81 40 32.2 trifuge for 5 minutes. Carefully remove the ether phase by Mean 86z rt 5 . 8 aspiration without removing any of the acid (aqueous) phase. N-Desmethyl diazepam 20 19.6 98 40 34.4 86 Wash the acid extract twice with 10 ml of ether, shaking for 60 57.2 95 10 minutes and centrifuging for 5 minutes, and remove the 57.2 95 60 ether by aspiration after each washing. [The sensitivity limits 80 81.6 102 of detection may be increased by extracting 2 to 4 rnl of blood 80 70.4 88 per assay. In this event use 6 ml of pH 9.0 buffer, and extract Mean 94% f. 6 . 0 twice with 15-ml portions of diethyl ether, respectively. The ether extracts are combined as before and backextracted with 5.0 ml of 2.0 N HCI. The acid layer has to be washed that are required as analytical standards are listed in Table I. 3 to 4 times with fresh 10-ml portions of ether to effect the Weigh out 10.00 mg each of the free base of medazepam (I) complete removal of the heavy layer of lipid material which is and its major metabolite., (PI to VII) into separate 10-ml present at the interphase.] volumetric flasks, dissolve in 1.0 ml of absolute ethanol and Make acid solution alkaline by slowly adding 5.5 ml of make up to volume with 20% acetone-hexane. These stock 2,ON NaOH. Check pH with indicator paper. This alkaline solutions (A) contain 1 rng/ml and are used to prepare subsolution is extracted twice with 10 rnl of ether, shaking for 10 acetone-hexane. Make sequent serial dilutions in 20 minutes and centrifuging for 5 minutes. The ether extracts serial 1 :10 dilutions of the respective solutions (A) to yield a are combined and evaporated to dryness in the water bath of solution (B) containing 1000 ngil0 p1 and a solution (C) cona Buchler Rotary Evaporator at 35-40 "G. The residues are taining 100 ng/lO pl, respectively, for each standard compound. vacuum dried for 30 minutes and dissolved in 100 pl of a 20 % Prepare the working standard solution (D) containing a acetone-hexane mixture, a suitable aliquot of which is anamixture of all the authentic standards as follows combining lyzed by GEC. the respective aliquots in a 10-ml volumetric flask and making Quantitation of Medazepam and Its Major Blood Metaboit up to volume with 2 0 x acetone-hexane solution, thus: lites. Because of the many metabolites present in blood in addition to the intact drug, the complex chromatogram of a Solution D blood ether extract has necessitated the use of the direct or Comp. I 0 . 2 ml of solution B 20 ng/lO pl absolute calibration technique. A suitable benzodiazepine/ I1 0 3 ml of solution B 30 ng/lO bl to 10 ml = 2-one reference standard which would elute with a retention 111 0 5 ml of solution C } with 20% 5 ng/lO pl time of about 15-20 minutes under these conditions has not acetone-hexane yet been found. This is the only retention area (free from IV 0 4 ml of solution C 4 ng/lO pl extracted impurities) in which a suitable reference standard 8 ng/lO p1 V 0 8 ml of solution C can be introduced for accurate quantitation. Although ComAliquots of solution D (1-10 pl) are injected into the chropounds 11and VI (Table I) were either absent or not recovered from blood in these studies they cannot be used reliably as matograph to establish the E.C. detector calibration curves and 100-pl aliquots are also added to blood as the internal reference standards because of their possible presence in any future situation. Consequently the direct calibration method standards for the determination of % recovery. Reagents. All reagents must be of analytical reagent grade has to be used using the external standard calibration curves (>99% purity) and all inorganic reagents were made up in Figure 3. distilled water (d.w.). The amount (ng) of medazepam and its major metabolites 1M H3BO3-Na2CO3-KC1buffer. Dissolve 61.8 grams of per aliquot of sample and recovered internal standard extracts boric acid (H3BO3)and 74.6 grams of KClil. of distilled water. injected is calculated directly from the external standard calibration curve (Figure 3). The recovery of each internal Dissolve 106 grams of Na2C03/1.of distilled water. To 630 ml of the boric acid-KCl solution, add 370 ml of the NazC03 standard is determined from the external standard curve solution to make a liter of buffer solution. Shake well and (Figure 3) as the ratio of ng recovered to ng added to blood, Table 11. The concentration of each component per ml of check pH, buffer it up to pH 9.0 if necessary with the Na2COs Table II. A. Recovery of W-Medazepam from Blood Determined by Scintillometry and EX.-GLC Gas Chromatography _ _ _ ~

x

x

'

1

1728

e


Ip:

Diozepam

P

n-desmelhyl Diazepam

PI PLI

11 I

21 E m , n .

RETENTION

TIME

( R t 1 IN MINUTES

3- hydroxy Diozepam

Oxozeoam

I

A

Figure 4. Gas chromatograms of diethyl ether extracts of ( A ) patient control urine; ( B ) control urine containing added authentic standards, and (C) patient urine (0-24 hr) post medication sample analyzed is calculated from the formula: ng of compound dilution (aliquot) factor ___ X Recovery factor of X 10-3 its Internal Standard 1

ml of sample analyzed

pg of compound/ml of sample

=

Assay in Urine. The major urinary metabolite is oxazepam VII present as a glucuronide conjugate. In addition measurable amounts of N-desmethyl diazepam V and N-methyl-3hydroxy diazepam VI are also present, the latter as a glucuronide conjugate. No measurable amounts of medazepam I, N-desmethyl medazepam 11, N-desmethyl I ,2-dehydro medazepam 111, or diazepam IV (see Figure 1, Table I) are present in urine (5). Since the major metabolites are present in the basic fraction as glucuronide conjugates, they can be extracted only after deconjugation by enzymatic hydrolysis with glucuronidase-sulfatase. The assay thus measures total urine concentrations of the different major metabolites present. Preparation of Standard Solutions. Use the stock solutions (A) and the serial dilutions (B) and (C) as prepared for use in the blood assay. Prepare a working solution (D') containing a mixture of the authentic standards as follows combining the respective aliquots in a 10-ml volumetric flask thus : Solution D' Comp. IV 0 . 4 ml of solution C \ 4 neilO ul V 0 . 8 ml of solution C to 10 ml = 8 ng/1o b1 with 20z acetone VI 0 . 5 ml of solution B hexane 50 ng/lO pul VI1 0 . 5 ml of solution C 5 ng/lO pl ~

I

One- to 10-pl aliquots of solution D' are injected into the gas chromatograph to establish the E.C. detector calibration curves and 100-pl aliquots are also added to urine as the internal standards for recovery determination.

Reagents. In addition to the reagents used in the blood assay, the following are also required. 1.0 molar phosphate buffer (pH 5.3). Dissolve 138.01 grams of NaH2P04.H20 per liter of distilled water. Titrate carefully with 1.ON NaOH to pH 5.3 and shake well by inversion to effect equilibration. Glusulase enzyme diagnostic reagent [lOO,OOO units glucuronidase and 50,000 units sulfatase /ml]. Endo Laboratories Inc., Garden City, New York. Procedure. Into a 50-ml Erlenmeyer flask, add 5.0 ml of urine, 5 ml of pH 5.3 phosphate buffer and 0.1 ml of glusulase (1% of total volume). Stopper loosely with cotton and place in a Dubnoff incubation shaker and incubate at 37 "C for 2 hours with mild shaking. Along with the samples, run a 5-ml specimen of control urine (taken preferably from the patient prior to medication) and duplicate 5-ml specimens of control urine containing 50 ng of oxazepam (VII), 40 ng of diazepam (IV), SO ng of N-desmethyl diazepam (V), and 500 ng of 3 hydroxy diazepam (VI) added as internal standards. After incubation and cooling, the solution is made alkaline (pH > 9) by adding 0.6 ml of 10N NaOH. This alkaline solution is quantitatively transferred into a 40-ml stoppered centrifuge tube and extracted twice with 10 ml of ether shaking for 10 minutes and centrifuging for 5 minutes. The ether extracts are combined and back-extracted with 5.0 ml of 6.ON HC1 by shaking for 10 minutes and centrifuging for 5 minutes. At this point follow the extraction procedure for medazepam as described for blood. The ether backwashed solution of 6.ON HC1 is made alkaline with 5.5 ml of 6.ON NaOW prior to extraction with diethyl ether. The final diethyl ether residue is vacuum dried and dissolved in 100 p1 of 20 % acetone-hexane, a suitable aliquot (5-10 pl) of which is analyzed by GLC. The sample may have to be further diluted because of the high amounts of oxazepam (VII) present. A typical chromatogram of the diethyl ether extract of urine is shown in Figure 4. Quantitation of the Major Urinary Metabolites of Medazepam. As in the blood assay, the presence of several metabolites in the urine results in a complex chromatogram of

ANALYTICAL CHEMISTRY, VOL. 42, NO. 14,DECEMBER 1970

0

1729

5

--Eilarnal

/o /;x

-Blood

Slonaavd

curve

AOCov~ry Curve

1

N-dtrme'hvl

dtorcpam

4

// //

3

//

// // //

/P

--Eilrrnal

2

x

-Blood

Standard Curve R*caverr

Curvr

I

D i o ~ r p a m Ip:

IL

0

0

I

4

a U

4 4 X

0.2

n

C

I

IO 20

I

I

1

20

30

40

40

60

Diazepam Ip

200 ng of Medazepam I

TOTAL CONCENTRATION I N N A N O G R A M S l o q l 1 1 0 0 & I

Figure 5. Standard curves of the electron capture detector response to varying concentrations of diazepam IV, and Ndesmethyl diazepam V using medazepam I as the reference standard the diethyl ether extracts of enzymatically hydrolyzed urine, Figure 4. Therefore the direct calibration technique has to be used as in the blood assay. The analysis of a 5- to 10-pl aliquot/100 MI of final solution usually results in a peak for oxazepam VI1 which is off scale whereas N-desmethyl diazepam V and 3-hydroxy diazepam VI are on-scale and can be quantitated. Further dilution is usually necessary to quantitate the peak due to oxazepam VI1 which is the major urinary metabolite present. No measurable amounts of medazepam I, N-desmethyl medazepam 11, N-desmethyl-l,2-dehydro medazepam 111, or diazepam IV are present in urine. It must be noted that although N-desmethyl medazepam I1 and oxazepam VI1 have identical retention times (6.0 min) under these GLC conditions, the high dilutions required for the quantitation of oxazepam VI1 eliminates any possible interference due to trace amounts of N-desmethyl medazepam I1 that may be present. The quantitation of each component in the chromatogram is calculated directly from the respective external standard curves (Figure 3), and the concentration of each component/ml of sample analyzed is calculated using the same formula as in the blood assay. Analysis for Diazepam and Its Major Metabolites in Blood and Urine. The metabolism of diazepam in man was elucidated by Schwartz et ul. (10). The drug was shown to undergo N-demethylation and hydroxylation to yield Ndesmethyl diazepam V which is the major blood metabolite, trace amounts of 3 hydroxy diazepam VI and oxazepam VI1 which is the major urinary metabolite. Thus diazepam produced in man as a metabolite following medazepam administration was also shown to undergo a similar metabolism, Figure 1 (5). The GLC assay described above for the determination of medazepam and its major metabolites in blood and urine, following the administration of Nobrium in man, is also applicable to the determination of diazepam and its major metabolites following the administration of Valium. The (10) M. A. Schwartz, B. A. Koechlin, E. Postma, S. Palmer, and G. Krol, J. Pharmacol. Exptl. Ther., 149, 423 (1965). 1730

a

assay is modified to include a reference standard which enables the use of the peak area ratio calibration technique for the quantitation of diazepam and its major metabolites in blood and urine. Ln this case medazepam I is used as the reference standard because it is quantitatively recovered and its GLC elution properties and E.C. detector response are compatible with those of diazepam and its metabolites. Preparation of Standard Solutions. Use the same stock solutions (A) and serial dilutions (B) and (C) for medazepam I, diazepam IV, N-desmethyl diazepam V, 3-hydroxy diazepam VI, and oxazepam VI1 as described previously. For the assay in blood; prepare mixtures of working standard solutions D* containing varying concentrations of diazepam (10-40 ng/O 1 ml) and N-desmethyl diazepam (20-80 ng/O. 1 ml) which also contains a constant amount of medazepam (200 ng/O.l ml) added as the reference compound. These solutions are made up conveniently in 10-ml volumetric flasks making up to volume with 20 % acetone-hexane solution. Ten-microliter (pl) aliquots of each of these solutions are injected in duplicate into the chromatograph to establish the E.C. detector external calibration curves, and 100-p1 aliquots are added to blood as the internal standards for the determination of recovery. Reagents. Same as in the blood assay described for medazepam. Procedure. ASSAYIN BLOOD. Into a 40-ml centrifuge tube, add 20 pl of standard solution (C) equivalent to 200 ng of medazepam (reference standard) and evaporate to dryness. Add 1.O ml of the blood specimen to be analyzed to this residue and dissolve by mixing, followed by 5 ml of pM 9.0 1M BOrate-KC1-NatCOa buffer and 10 ml of diethyl ether. Along with these unknowns, process 1.0 ml of control blood (preferably taken from the patient prior to medication or from a pooled control source) and four separate 1.0-ml aliquots of control blood added to the residue of 100 pl of the working standard solutions D* covering the concentration of diazepam (10-40 ng), N-desmethyl diazepam (20-80 ng) and 200 ng of medazepam as the reference standard. These samples are used to determine the blood recovery curve (Figure 5 ) which is used for the quantitation of the unknowns.


The samples are then processed exactly as described for medazepam, the final residues vacuum dried and dissolved in 100 p1 of 20% acetone-hexane, a suitable aliquot (1 to 10 p1) of which is analyzed by E.C.-GLC. The peaks due to medazepam, diazepam, and N-desmethyl diazepam are identified by their respective retention times (Figure 21, and their respective peak areas determined as before using the slopebase line technique. Calculation. The peak area ratios of diazepam to medazepam and N-desmethyl diazepam to medazepam in the recovered internal standards is determined and plotted graphically us. concentration as shown in Figure 5 to establish the blood recovery curve. The peak area ratios of diazepam, and N-desmethyl diazepam to medazepam in the aliquots of the unknowns injected is determined similarly. The concentration of the two components in the unknown represented by their respective peak area ratios is read directly from the blood recovery or internal standard curve. Since the peak area response ratios of diazepam or its metabolite to medazepam in any given sample is constant irrespective of the actual volume of sample injected, the concentration in the unknowns is extrapolated directly from the internal standard curve (Figure 5). Since the internal standards and unknowns are all dissolved in 100 pl of final solution, there is no dilution or aliquot factor to be considered unless further dilution of the unknowns is necessary. The recovery factor for both internal and reference standards remains constant throughout and is not needed for the calculation of the unknown. Thus: Concn (ng) in Unknown (determined from int. std. curve) X ml of sample assayed dilution factor 10-3 = pg of diazepam or N-desmethyl diazepam per ml of blood

where: concn (ng) = amount in the aliquot of sample extract injected, extrapolated from the internal standard curve. Dilution (aliquot) factor = sample aliquot injected corrected for total volume if further dilution (> 100 p l ) of the sample is necessary. =

conversion factor for nanograms to micrograms.

Determination of % Recovery: The use of a reference standard (medazepam) in the analysis together with the internal standards (diazepam and N-desmethyl diazepam) added to blood eliminates the need to determine the actual % recovery obtained with each run. Once the actual recovery factor has been experimentally determined by GLC (ciz., = 86% =t6.0 for diazepam and 94z i. 6.0 for N-desmethyl diazepam) this factor can be assumed to be constant through-

out. Since the overall recovery of each compound is of the same order of magnitude, any change in the overall recovery of one would be automatically reflected in the recovery of the other to the same degree. Hence, irrespective of the actual recovery of each, the response ratio of the E.C. detector would be constant and thus compensate for any variations incurred in sample processing. Consequently the response ratio of diazepam or N-desmethyl diazepam to medazepam in the recovered internal standards and in the unknowns would represent the actual amounts of each compound present irrespective of the recovery. Calculation of % Recovery of Internal Standards. The peak area response ratio of the recovered internal standards is plotted against concentration of internal standards (as shown in Figure 5) to construct a blood recovery standard curve. The slope Qf this curve is compared directly against that of the external standard curve (Figure 5 ) to obtain the overall % recovery. Alternatively, the peak area response ratio of any recovered internal standard is compared directly against

that of a corresponding concentration of the external standard thus : [Peak Area Response Ratio] Int. Std. X 100 [Peak Area Response Ratio] Ext. Std.

=

%Recovery

The sensitivity of the GLC method is of the order of 0.01-0.02 pg of diazepam and 0.02-0.04 pg of N-desmethyl diazepam per ml of blood. The overall recovery is of the order of 86 % rt 6.0 for diazepam and 94% i 6.0 for N-desmethyl diazepam respectively, Table 11-B. Assay in Urine. The major urinary metabolite of diazepam is the oxazepam-glucuronide. In addition small amounts of 3-hydroxy diazepam-glucuronide and N-desmethyl diazepam are present. No measurable amounts of diazepam are seen in urine (IO). The glucuronides have to be deconjugated by enzymatic hydrolysis at p H 5.3 prior to extraction into diethyl ether at an alkaline pH. The sample preparation, extraction, and GLC analysis is carried out exactly as described in the urine assay for medazepam and its metabolites. Although medazepam can be used as the reference standard in this assay, it is not practical because of the high dilution factor necessary for the quantitation of oxazepam compared to that required for the minor components N-desmethyl diazepam and 3-hydroxy diazepam. Therefore, the direct calibration technique has to be used for the quantitation of the urinary metabolites as described in the medazepam urine assay. Previous studies by Schwartz et a/. (IO)utilizing 3H labeled diazepam showed that maximum deconjugation (approx. 80 %) occurred under the conditions described. Therefore, it is assumed that a similar efficiency in the deconjugation reaction is obtained in these studies. The overall recovery of the major urinary metabolites oxazepam, N-desmethyl diazepam, and %hydroxy diazepam when added to urine as authentic reference standards ranged from 80-85 %. The sensitivity limits of the assay are of the order of 0.02-0.04 pg of each compound/ml of urine. RESULTS AND DrsCussIorv

Medazepam and diazepam undergo extensive biolransformation in man and in the dog resulting in the presence of several components measurable in both blood and urine (5, IO). This necessitates selective extraction and chromatographic separation prior to quantitation. The parent drugs and their respective biotransformation products are all weakly basic compounds which can be quantitatively extracted into diethyl ether from biological materials buffered to pH 7.0 or greater. The recovery of medazepam, diazepam, and their major metabolites added to blood buffered to pH 7.0 and pH 9.0, extracted into diethyl ether and back-extracted into 2.ON MC1 was determined by the ultraviolet (UV) absorption spectra of these compounds in acid. The compounds were shown to be quantitatively extracted above pW 7.0, but the ether extract of blood buffered to pW 9 with borate buffer gave cleaner control chromatograms and was preferred. Diazepam, N-desmethyl diazepam, %hydroxy diazepam, and oxazepam exhibit their main UV absorption maxima around 240-243 mp with a secondary peak at 285-287 mp, whereas medazepam (255 mp) and N-desmethyl medazepam (245255 mp) have dissimilar spectra. The compound, N-desmethyl dehydro medazepam 111 undergoes partial hydrolysis and rearrangement in 2.074 acid (HCI) and on exposure to light in ethanol to yield the benzophenone ACB VI11 and several unidentified products as indicated by thin-layer chromatographic (TLC) analysis. The UV spectrum of III in ethanol showed a maximum at 225 mp with st second smaller peak at 340 mp whereas in ethanolic-0.1N HCl(1 :I), the maxima had shifted to 240 mp and 420 mp, respectively,


a

1731

indicating a chemical change. The UV spectrum resembled that of the benzophenone (ACB) which exhibits maxima at 238 mp and 395 mp, respectively. However, no intact compound I11 and only small amounts of the benzophenone were recovered from the acidic solution after the addition of alkali and extracting it into diethyl ether. This compound is therefore not extracted as the intact moiety and cannot be quantitated by this method. The concentration of HC1 used in the back-extraction of diethyl ether is critical to the stability of benzodiazepines. Medazepam and its major blood metabolites are stable in 2N HCl, and are recovered quantitatively as the intact moiety upon neutralization and ether extraction. The major urinary metabolites 3-hydroxy diazepam VI and oxazepam VI1 require 6N HC1 for quantitative back-extraction from diethyl ether. These facts were verified by thin-layer chromatography (TLC) of the diethyl ether extracts and either UV or GLC analysis of the intact compound after elution. In 4 or 6N HC1 or concd H2S04however, medazepam, (at room temperature) undergoes partial decomposition yielding measurable amounts of the N-l-methyl-2-quinazolinone according to the following scheme:

,,,/, Oxidative

C1

C=N

I earra ngernent

I

Quinazolinone derivative

Diazepam, N-desmethyl diazepam, 3-hydroxy diazepam, and oxazepam undergo similar rearrangement in concd H2SOato yield their respective fluorescent quinazolinones. The Ndesmethyl compounds produce stronger fluorescence than the N-methyl analogs, probably because of accelerated reaction rates and higher yields of their quinazolinones. The synthesis of these quinazolinones by the oxidation of their respective benzodiazepine or -2-ones was reported (11). The products recovered from these strong acid solutions from medazepam showed a doublet on GLC and two distinct spots on TLC analysis; the major one being the parent compound, and the minor one being the quinazolinone. The quinazolinones are strongly fluorescent on silica gel GIF chromatoplates when viewed under long wave UV (3660 A). They also fluoresce strongly in concentrated H2S04at room temperature with activation at 420 mp and emission at 505 mp. Diazepam(1V) and its major metabolites (V, VI, VII) are all stable in 2, 4, 6N HC1 at room temperature. However, they undergo hydrolysis when heated at 100 O C for 1 hour in (11) A. M. Felix, J. V. Earley, R. I. Fryer, and L. H. Sternbach, J. Heterocycl. Chem., 5 , 731 (1968). 1732

6N HC1 to produce quantitative yields of their respective benzophenones MACB and ACB (7, 8). The extraction scheme used in the assay uses 2N HCl to ensure the chemical stability of the several benzodiazepines, and -2-ones present, resulting in the quantitative recovery of all the major metabolites of medazepam and diazepam, respectively. The only exceptions are the three benzophenones MACB, ACB, and ACHB, which if present in blood or urine as metabolites are not back-extracted into 2N HC1 from the respective diethyl ether extracts, and N-desmethyl-1,2-dehydro medazepam I11 which is chemically unstable in acid. These were found to be only very minor constituents of blood and urine (5); hence their loss is not significant. In the investigation of suitable analytical parameters for the GLC analysis of these benzodiazepines, several factors of significance became apparent. Although the methyl silicone OV-1 was reported to be a satisfactory phase for the analysis of several benzodiazepines using the flame ionization detector (9), the resolution of the various compounds from a biological extract was not complete, resulting in overlapping peaks and poor sensitivity using the E.C. detector. The simplified procedure for sample preparation reported by these authors (9) was inapplicable to GLC analysis with the E.C. detector because of the impurities present in the sample extract. The sample extraction and clean-up procedures required for this assay were derived from the published method (7,8) except that no acid hydrolysis is involved; hence the recovery of the various components as the intact benzodiazepines and -2one compounds. The response characteristics of the “3Ni E.C. detector (pin cup design) operated in the pulsed dc mode has to be carefully determined for each compound with respect to the voltage applied, the pulse rate, and pulse width used. Changing any one of the parameters affects not only the response with respect to sensitivity and linearity but also the peak shape. The detector parameters used for the quantitation of several compounds in a single operation has of necessity to be the best compromise for all the compounds involved with respect to sensitivity, linearity, reproducibility, and peak shape (12). Since E.C. detector response to a compound is governed by several factors such as the electron affinity or capture coefficient, which varies with the type of compound in question, the detector geometry (e.g., parallel plate, cylindrical foil, pin cup, on photo ionization), the mode of operation (dc us. pulsed dc), and the type and flow rate of carrier gas required (nitrogen us. argon methane), all these factors have to be evaluated with respect to each compound to be analyzed to arrive at the best combination of parameters (13-15). Because of the difference in response to different compounds and the narrow linear dynamic range of E.C. detection, the concentration range in which quantitative measurements can be made is limited. It is also apparent that the sensitivity and range of linearity for the various compounds will differ, as seen in Figure 3. The parameters established for the chromatographic separation and quantitation by E.C. detection, enables the complete resolution and quantitation of the major blood and urinary components following medazepam administration. (12) W. L. Yanger, L. M. Addison, and R. K. Stevens, J . Ass. Ofic. Anal. Chem., 49, 1053 (1966). (13) W. E. Wentworth, E. Chen, and J. E. LovelockJ. Plzys. Chem., 70, 445 (1966). (14) J. E. Lovelock, “Gas Chromatography,” 1968 Ed., C. L. A. Harbourn, The Institute of Petroleum, London. (15) P. Devaux and G. GuichonJ. Chromatogr. Sei., 7, 561 (1969).


The chromatograms of the diethyl ether extracts of blood, Figure 2, and of urine, Figure 4, show the base-line resolution of all the components enabling accurate quantitation of each compound. Either the direct calibration techniques using peak areas as in the case of medazepam and its metabolites or the relative calibration technique using peak area ratios with respect to an internally added marker or reference standard as in the assay for diazepam and its metabolites can be used. The use of medazepam as the reference marker in the assay for diazepam has led to the automation of this assay in our laboratories using the dry injection technique of sample introduction on wire gauzes, (Barber-Colman Chromatographic System). The search for a suitable reference standard for the medazepam assay has not yet been satisfactorily concluded. The assay for urine yields the total diethyl ether extractable components after incubation with glucuronidase/sulfatase enzyme. The major compound is oxazepam-glucuronide with smaller amounts of the 3-hydroxy diazepam glucuronide and N-desmethyl diazepam. Trace amounts of N-desmethyl medazepam which may be present cannot be directly quantitated since it is not resolved from oxazepam. This does not cause any interference in the assay for oxazepam due to the high dilution factors needed to quantitate the compound in its linear range. The long retention time of 22 minutes required for the elution of 3-hydroxy diazepam and its poor response to the E.C. detector are a disadvantage, although it is completely resolved from N-desmethyl diazepam (R1= 12 minutes) Figure 4. This situation can be corrected by preparing the trimethyl silyl (TMS) derivatives of 3hydroxy diazepam and oxazepam as follows : CH i

3-Hydroxy diazepam

I

/

TMS derivatives

The oxazepam-TMS derivative shows no change in its sensitivity to the E.C. detector, and elutes at the same retention time as before (6 minutes). The peak does, however, show a small

increase in tailing contrary to expectations, probably due to an increase in the polarity of the -NH group in the benzodiazepin ring due to the electron inductive effects of the TMS group, The 3-hydroxy diazepam-TMS derivative, however, shows a significant increase in sensitivity (approx. 10-fold) to the E.C. detector, and also elutes as a sharp Gaussian shaped peak at approximately half its former retention time. The shortened retention time of about 12.8 minutes coincides however with that of N-desmethyl diazepam, consequently interfering with the quantitation of this metabolite. These difficulties can be effectively solved by employing a differential extraction procedure for urine as follows. Diethyl ether extraction of urine buffered to pH 9.0 will quantitatively remove any medazepam, N-desmethyl medazepam, diazepam, and Ndesmethyl diazepam which can then be processed and quantitated according to the blood assay for rnedazeparn. The urine is then acidified to pH 5.3, incubated with glusulase, titrated back to pH 9.0, extracted into diethyl ether, and processed as per original urine GLC assay. The final residue (after clean-up) will contain only the deconjugated metabolites; 3-hydroxy diazepam and oxazepam. This residue is then vacuum dried to remove any moisture, dissolved in 250 pl of diethyl ether to which is added successively 40 pl of hexamethyl disilazane (HMDS) and 20 pl of trimethylchlorosilane (TMCS), the tube stoppered and mixed vigorously for 1 minute on a vortex action high speed mixer (16). The silylation reaction is complete in 15 minutes at room temperature. The samples are evaporated under dry nitrogen to remove all the reagents, vacuum dried again for 15 minutes, and dissolved in 100 pl of 20% acetone-hexane solution, an aliquot of which is analyzed by GLC. The chromatogram will now show two peaks representing the TMS derivatives of the parent compounds. The procedure is applicable to the quantitation of urinary metabolites following the administration of both medazepam and diazepam. Because the technique is quite time consuming, and the amounts of the directly extractable metabolites being small, this procedure was not developed as a routine assay. Application of the Method to Biological Specimen. The blood level curves and urinary excretion of medazepam and its major metabolites were determined in two human subjects following the administration of single and multiple oral doses of Nobriym. Single Oral Doses. A female subject (M. F.) was given a single oral dose of 50 mg (5 X 10 mg tablets) and blood specimens were collected prior to medication and thereafter at 0.5, 1, 2, 4, 8, 12, and 24 hours post medication. The blood level curve is shown in Figure 6. The curve indicates rapid oral absorption of the drug with a peak blood level of 0.98 pg/ml being attained 1 hour after medication. The levels declined rapidly to low levels of 0.02 to 0.03 pg/ml measurable up to 24 hours. Measurable levels of diazepam produced as a metabolite ranging from 0.02 to 0.04 pg/ml were also seen from 30 minutes to 24 hours post medication. Trace amounts of N-desmethyl diazepam were noted but were not quantitated. The analysis of the urines revealed that no detectable amounts of medazepam, N-desmethyl medazepam, diazepam, and N-desmethyl diazepam were excreted. The major recoverable metabolite appeared to be oxazepam, the GLC quantitation of which was not possible because of interference from quinidine and/or its metabolites which were present as a result of recent medication. (16) J. A. F. de Silva, N. Munno, and N. Strojny, J. Pharrn. Sci., 59, 201 (1970).


1733

Potient wt.

Dose

Figure 6. Blood level fall-off curves of medazepam and its metabolite diazepam following a single 50mg oral dose of Nobrium

M.F. ( f ) 77k9 5 x l O r n g Tablers(NOBRIUM@ 1

0

0

2

m

DIAZEPAM

0 01 0 1 2

4

8

12

24

HOURS POST ADMINISTRATION

Multiple Dose Administration. A male subject (H. R.) was given single oral doses of 50 mg (5 x 10 mg tablets) for four consecutive days, each dose being given 24 hours apart. Blood specimens were collected prior to medication and thereafter at 0.5, 1, 2, 4, 8, 12, 24, 25, 48, 49, 72, 73, and 96 hours post medication. Urines were collected in 24-hour pools over the 96-hour experimental period. The blood level curves of medazepam and its major metabolites are shown in Figure 7. Measurable levels of medazepam and its major metabolites, diazepam and N-desmethyl diazepam were first

seen 1 hour after the initial 50-mg dose. A peak blood level of 0.37 pg medazepam per ml was seen at 2 hours and declined rapidly to a level of 0.07 pg/ml which was maintained between 12 and 24 hours. During the same 24-hour period, the build-up of diazepam (peak of 0.08 pg/ml at 2 hours) and its decline almost paralleled that of medazepam. On the other hand N-desmethyl diazepam showed a steady increase with a peak level of 0.13 pg/ml at 4 hours, and was almost constant between 8 to 24 hours at 0.10 ,ug/ml. Following the second through the fourth doses, the intact drug was mea-

Dose 5 0 mg ( 5 x IO m9 Toblels l o t orrow

--___-

-----

0 Medorepom I

0 4 8 1 2

24

48

72

96

HOURS POST A D M I N I S T R A T I O N

Figure 7. Blood level curves of medazepam and its major metabolites in man following multiple oral doses of Nobrium 1734


SYSTEM

f :

90' 10

'i

,$c

-_

.+

9 0 . IO

-

B

[w., l i

;to

Blood E x l r o c l r

0 5 I o 96

.r

hrS

2 6 r l Pooled

1..

..

bo

'bo

II

SYSTEM

c C h l o r Acetone

0

A

I+-

I

I

U

c C h l o i . Acelow

L

Uiine Ertroclr 0 l o 96 h r s 2 0 m l Pooled

I -

(...., II

..

'._.I

.,.*. ..

I,

:. * ,: SYSTEM Rf

I

I ~ 0 4 5

n :o.12 m:o33 1P.038 P -020 Yl'O29 pn=oo9

SYSTEM Rf

1

3

II

SYSTEM

045

Rf I

I

SYSTEM

049 TT 0.12 m*040 TP.043 Pi023 PI2030 3LII. 0 I1

n *0 12 032 0.41 P.021 m: 0 3 4 HI: 0 0 8 I p s

I I

R f I ? 0.42 0.12

a

n= m: 0.30

IP: 0.39 P =0.16

m -0.33 PII. 0.08

Figure 8. Two dimensional thin-layer chromatograms of the acetone-hexane solutions after GLC analysis of the diethyl ether extracts of ( A )blood and ( B )urine surable 1 hour post dosing with reproducible levels ranging from 0.38 to 0.58 pg/ml being attained, the levels declining however to nonmeasurable amounts in 24 hours. The levels of diazepam on the other hand remained fairly constant ranging from 0.05 to 0.07 pg/ml during the 24- to 96-hour period. The levels of N-desmethyl diazepam however, showed a tendency toward accumulation, the levels increasing steadily from 0.11 pg/ml at 24 hours to a level of 0.45 pg/ml at 96 hours. In addition measurable amounts of N-desmethyl medazepam ranging from 0.11 to 0.22 pg/ml were seen 1 hour after each consecutive dose at 24,48, and 72 hours. The urinary excretion data on this subject are given in Table 111. The major urinary metabolite was oxazepam which was excreted in increasing amounts with time up to 72 hours. The total amount recovered was approximately 5.4 mg (calculated as its medazepam equivalent) representing 2.7 of the total dose (200 mg). Smaller amounts of N-desmethyl diazepam and 3 hydroxy diazepam were excreted, together representing only 0.6% of the total dose. No measurable amounts of medazepam, N-desmethyl medazepam, or diazepam were seen. Characterization of the Blood and Urinary Metabolites by Thin-Layer Chromatography. Two-dimensional, thin-layer chromatography (TLC) was used to further characterize medazepam and its metabolites seen in blood and urine. The acetone-hexane final solutions of the diethyl ether extracts of blood and urine from subject (H. R.) on multiple doses, remaining after GLC analysis, were pooled, concentrated by evaporation and transferred onto Brinkmann [FZS4] precoated silica gel G / F chromatoplates, The plates were developed in vapor saturated tanks lined with Whatman No. 1 paper in two dimensions using chloroform :heptane :ethanol (10 : 1 O : l ) in the first dimension, and ch1oroform:acetone (90: 10) in the second dimension. Authentic standards of all the compounds expected to be present were run as markers along the margins (Figure 8). The chromatogram of the pooled blood specimen (Figure 8-A) representing 26 ml showed the presence of only med-

Table 111. Urinary Excretion Data in Man Following Multiple Oral Doses of Medazepam (Nobrium) Total (pg) recovered, per volume voided N-

Time period, Volume Desmethyl 3-Hydroxy Oxazepam VI1 hr voided, ml diazepam V diazepam VI 0-24 24-48 48-72 72-96

4025 3225 3410 2380

81 194 205 190 670

81 65 136 167 449

483 1226 2217 1785 5711

Total (pg) recovered = p g equivalents of medazepam = 670 404 5391 Medazepam equivalent recovered = 6.465 mg. Total dose Total dose recovered = 3.23. administered (50 X 4) = 200 mg. azepam I, N-desmethyl medazepam 11, diazepam IV, and N-desmethyl diazepam V as dark UV absorbing spots when viewed under a short wave UV lamp, thus confirming the components seen by GLC. The plate was left exposed to the atmosphere and to light overnight and reexamined under long wave UV. The spots corresponding to medazepam and Ndesmethyl diazepam V showed an intense blue-green fluorescence due to the photochemical oxidation of these compounds to their respective quinazolinones. This reaction can be used as a toxicological diagnostic test for medazepam. The chromatogram of the pooled urine specimen (Figure 8-B) representing 20 ml showed the presence of small amounts of N-desmethyl diazepam V, 3-hydroxy diazepam VI and what appeared to be traces of N-desmethyl medazepam TI, and diazepam IV. The major component was oxazepam VI1 again confirming the GLC findings. A diagnostic test for medazepam and its metabolites, diazepam, N-desmethyl diazepam, 3 hydroxy diazepam, and oxazepam when present in microgram amounts on a TLC plate is the finding that spraying the plate with 10% HzSO~ produces a characteristic yellow to blue-green fluorescence


*

1735

which is observed under longwave UV. The fluorescence is attributed to the formation of the respective quinazolinones of these compounds. Medazepam, N-desmethyl diazepam, and oxazepam all show strong fluorescence whereas diazepam and 3-hydroxy diazepam fluoresce weakly, while N-desmethyl medazepam does not fluoresce at all. These compounds can be further characterized by acid hydrolysis in situ in a 105 “C oven when the N-methyl and Ndesmethyl 1,4-benzodiazepin-2-0nesare converted into their respective yellow colored benzophenones MACB and ACB. Those compounds which hydrolyze to the ACB derivative give a pink to purple colored spot of the diazo-chromophore when reacted with the Bratton-Marshall reagents (17), characteristic of the primary aromatic amine group.

The results of these experiments using the pharmaceutical trade formulation of medazepam (Nobrium) in man are in agreement with the findings of Schwartz and Carbone (5)who used ‘Gmedazepam administered to man for their metabolic studies.

(17) A. C . Bratton and E. K. Marshall, J. Biol. Chern., 128, 537 (1939).

RECEIVED for review June 1, 1970. Accepted September 8, 1970.

ACKNOWLEDGMENT

The authors wish to thank Dr. A. S. Leon and his staff for conducting the clinical studies and for the supervision of the human subjects at the Beth Israel Hospital, Newark, N. J. We also thank Messrs. T. Danielian and R. Mc Glynn for the drawings of the figures presented.

Quantitative Analysis of Triglyceride Mixtures by Mass Spectrometry Ronald A. Hitesl Northern Regional Research Laboratory, Peoria, Ill. 61604 A rapid and sensitive method has been developed for determining the molecular weight distribution of triglyceride mixtures that occur naturally as fats and oils. The mass spectrum of the fat is measured by placing the sample directly into the ion source, and the consequent fractionation of the sample, caused by molecular distillation, is corrected. Triglyceride compositions have been measured for kokum and cocoa butters, olive, peanut, cottonseed, corn, soybean, sunflower, safflower, and linseed oils. When observed values were compared to theoretical values, they had a high overall correlation. Several potential applications of this mass spectral technique to problems of lipid research have been tested. A NATURAL FAT may consist of several dozen different triglycerides. For example, Table I lists the 18 possible triglycerides formed from three fatty acids-palmitic, stearic, and oleic. Although the amount of each triglyceride occurring in a fat has considerable academic interest and practical significance, there are few methods for the quantitative analysis of triglyceride mixtures. Such procedures as fractional crystallization, ester distillation, countercurrent distribution, thin-layer chromatography, and gas chromatography either are time consuming, lack resolution, or require large samples (I). To overcome some of these problems and to provide an analytical technique suitable for research and industrial applications, a semiautomated mass spectrometric method was developed for measuring the triglyceride composition of fats. The method is both Present address, Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Mass. 02139 (1) M. H. Coleman, Aduan. Lipid Res., 1, 1 (1963); H. J. Dutton, “Progress in the Chemistry of Fats and Other Lipids,” Vol. VI, R.T. Holman, W. 0. Lundberg, and T. Malkin, Ed., Macmillan, New York, 1963,p 313. 1736

rapid (ca. ‘12 hour per sample) and sensitive (less than 100 pg of fat is needed) and involves no pretreatment of the sample. Good mass spectra of triglycerides can be obtained by introducing the sample directly into the ion source (2). Molecular ions, M+, and ions due to the loss of 18 amu from M+, (M - 18)+, are observed. Other features of these spectra have been reported elsewhere (2). Since there are no intense peaks within at least 200 amu of the molecular ion to act as interferences, it was predicted that the molecular weight distribution of a triglyceride mixture could be simply measured from its mass spectrum. This prediction has now been verified experimentally. Because molecular weight gives only the number of carbon atoms and double bonds in the triglyceride, it is impossible to distinguish between positional isomers (PSO us. POS us. OPS ; see Table I for explanation of abbreviations) or between isologs (dioleylstearin us. distearyllinolein). This limitation reduces the number of components that can be differentiated. In Table I, for example, the 18 possible triglycerides are grouped into 10 discrete molecular weights. EXPERIMENTAL

Materials. Tristearin, 1,3-distearylpalmitin, 1,3-dipalmitoylstearin, and tripalmitin were the gift of W. F. Geddes, University of Minnesota. Estimated purity is better than 98%. 1,2-Dipalmitoylolein was used as purchased from Supelco, Inc. (purity 99+%). Kokum butter was supplied by E. S . Lutton, Procter & Gamble. The other vegetable oils came from commercial sources. None of these oils were refined or treated in a way that would alter the triglyceride composition; all were stored at less than 5 OC. The modified tallow samples came from an industrial source. An artificial (2) M Barber, T.0. Merren, and W. Kelly, Tetrahedron Lett., 1063 (1964).


Determination medazepam (Nobrium), diazepam (Valium), and their

Recommend Documents