CHIRALITY 7 158-162 (1995)

Direct Separation of Albuterol Enantiomers in Biological Fluids and Pharmaceutical Formulations

Using a,-Acid Glycoprotein and Pirkle Urea Type Columns

HASSAN Y. ABOUL-ENEIN, AND VINCE SERIGNESE Bwanalytical and Drug Development Laboratoty, Biological and Medical Research Deparhnent, King Faisal

Speaklist Hospital and Research Centre, Riyadh, Kingdom of Saudi Arabia

ABSTRACT A direct, isocratic, and simple chromatographic method is described for the resolution of racemic albuterol using the al-acid glycoprotein chiral stationary phase (AGP- CSP) under reverse phase conditions. The effect of various organic modifiers, temperature, and phosphate buffer ionic strength on the separation factor (a) and stereochemical resolu- tion factor (R,) has been studied. The enantiomeric separation of albuterol was also achieved using a urea-type CSP of (S)-indoline-2-carboxylic acid and (R)-1-(a-naphthyl)ethylamine, known as Chirex 3022, running in the normal phase mode. The effect of different organic acids added to the mobile phase was examined and the chiral recognition mechanism(s) is discussed. Solid phase extraction with c18 Sep-Pak cartridges was applied as a clean-up step to determine the enantiomeric ratio between (-)-R and (+)-S-albuterol in pharmaceutical formulations and in human plasma. o 1995 Wiley-Liss, Inc.

KEY WORDS: albuterol enantiomers, salbutamol enantiomers, a,-acid glycoprotein, Pirkle urea type column, chiral recognition mechanisms(s), chiral analysis

Albuterol, also known as salbutamol[2-(tert-butylamino)-l- (4-hydroxy-3-hydroxyethylphenyl) ethanol], is a selective P,-adrenoceptor agonist widely used in the treatment of bron- chial asthma’ and to relieve bronchospasm in patients with reversible obstructive airway diseases. ~4 Like several other drugs of this class, albuterol is administered as a racemic mixture of (+)-S- and (-)-R-enantiomers (Fig. l), although its pharmacological effect resides in the (- )-R-enantiomer.

There are several reports indicating that (+ )-S-albuterol induces airway hyperreactivity to leukotriene C4 and may cause adverse effects in asthmatic patients, possibly leading to Recently, Galland and Blackmans confirmed the lindings of Morley and co-workers7 that the non-bronchodila- tor (+)-S-albuterol enhances airway reactivity. Furthermore, administration of racemic albuterol also increases airway reac- tivity to histamine. In light of contemporary clinical data which suggest that regular daily long-term use of &-agofists such as albuterol has been shown to worsen the diseaseg and to be associated with an increase in asthma mortality, lo the pharma- cology of (+)-S-albuterol and other (+)-&-agonist enanti- omers merits clinical investigation and warrants the “racemic switch.” Use of the pure (-)-R-albuterol enantiomer should thus be considered instead of the currently used racemic mixture.

Methods of analysis of albuterol have been reviewed by Aboul-Enein et al. l1 and Kucharczyk and Segelman. l2 Several HPLC procedures for the determination of racemic albuterol have been reported. 13-17 He and Stewart18 reported an HPLC 0 1995 Wiley-Liss, Inc.

assay for albuterol enantiomers in human serum after chemi- cal derivatization with 2, 3, 4, 6-tetra-0-acetyl-a-glucopyra- nosy1 isothiocyanate. The diastereoisomeric thioureas of al- buterol were analyzed on a C18-reverse phase column. Recently, Adams and Stewartlg developed a chiral HPLC assay for the albuterol enantiomers in human serum without derivatization. Chiral separation was accomplished under nor- mal phase conditions on a Sumichiral OA 4700 column.

This paper describes the resolution of racemic albuterol using an al-acid glycoprotein (AGP) column under reverse phase conditions, and a Pirkle urea type chiral stationary (CSP) of (S)-indoline-2-carboxylic acid and (R)-1-(a-naphthyl) ethylamine (known as Chirex 3022) (Fig. 21, under normal phase conditions.

Both methods may be applied to determine the enantio- meric ratio of albuterol enantiomers in pharmaceutical dosage forms and human plasma after solid phase extraction with a

Sep-Pak cartridge.

Received for publication October 18, 1994; accepted November 27, 1994. Presented at the 5th International Symposium on Chiral Discrimination, Stock- holm, Sweden, 25-28 September 1994. Address reprint requests to Professor Hassan Y. Aboul-Enein, Bioanalytical and Drug Development Laboratory, Biological and Medical Research (MBC- 03), King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211, Saudi Arabia.

DIRECT SEPARATION OF ALBUTEROL ENANTIOMERS 159

CH2OH H CH OH OH

HO&g-CH2-NH C(CH3)s HO-&E-CH2-NH-C(CH3)3 - - OH H

(+)-S-Albuterol (-)-R-Albuterol

Fig. 1. Absolute configuration of (+)-S- and (-)-R-albuterol.

Fig. 2. The structure of the CSP-Chirex 3022 made of (S)-indoline-2-carbylic acid linked to (R)-l-(cr-naphthyl)ethylamine urea.

MATERIALS AND METHODS Reagents and Materials

Injectable racemic albuterol sulfate, in lml ampoules (0.5 mg, lot # B0781EA) was obtained from Allen & Hanburys Ltd. (Greenford, England). The enantiomers (+)-S-albuterol acetate (Lot# AH6619H) and (-)-R-albuterol acetate (Lot# AH6620H) were a generous gdt from Glaxo Research Insti- tute, Inc. (Research Triangle Park, North Carolina). HPLC grade trichloroacetic acid, 1, 2-dichloroethane, trifluoroacetic acid, hexane, acetic acid, formic acid, potassium dihydrogen phosphate, l-propanol, 2-propanol, ethanol, methanol, and acetonitrile were purchased from Fisher Scientific (Spring- field, NJ). CI8 Sep-Pak cartridges were purchased from Wa- ters Associates (Millford, MA).

Chromatography The chromatographic system, purchased from Waters,

consisted of a 990+ photodiode array detector, with a NEC 286 computer, including a black and white monitor, keyboard, and accompanying 990+ software, a M45 pump, a U6K injec- tor and a 5200 printer/plotter. The Chiral-AGP column (100 mm x 4 mm ID, 5 p.m particle) was purchased from Chrom Tech AB (Hagersten, Sweden). The Chirex 3022 (3-ICA (R) NEA column (250 x 4 mm ID, serial # 62923) was a gdt from Phenomenex (Torrance, CA). Various mobile phases as well as the chromatographic conditions used are described under “Results and discussion.”

Plasma Sample Preparation and Solid Phase Extraction

Pooled human plasma was obtained from the King Faisal Specialist Hospital Blood Bank (Riyadh, Saudi Arabia). Inject- able racemic albuterol sulfate was diluted 10-fold in a total volume of 2 ml with phosphate buffer. The h a l pH was 7.3. The contents were added to 1 ml of blank plasma and vortexed for 3 min. The sample was extracted using a solid phase C18 cartridge according to He and Stewartla with the following modifications: Following sample application, the cartridge was washed with 1 ml phosphate buffer (pH 7.3),2 ml water, and 2

(min) o 5 10 15

Fig. 3. Enantiomeric separation of racemic albuterol sulfate. Column: a,-acid glycoprotein (100 x 4 mm id.) mobile phase: 0.01 M phosphate buffer, 0.13%, v/v, 2-propan01, pH 7.5; flow rate: 0.5 ml/min; chart speed 0.5 d m i q temperature: 23°C detector: UV 276 nm; sample quantity: 3.5 moles.

ml 15% methanol in acetonitrile. Albuterol sulfate was eluted with 1 ml methanol and directly injected for HPLC analysis. Human plasma collected from a healthy subject receiving race- mic albuterol orally was processed as described above.

Chromatographic Parameters Capacity factors (k’) were calculated using the equation

k’ = (VR - V,)/V, where V, is the elution volume and V, is the void volume. The separation factor (a) was calculated using the equation a = k;/k i where k ; and k i are the capacity factors for the iirst and second eluted peaks.

RESULTS AND DISCUSSION Figure 3 shows a chromatogram of the resolution of al-

buterol enantiomers (80% baseline separation) on the AGP column at optimum chromatographic conditions. The enantio- meric elution order was determined by chromatographing the individual enantiomers, using the reverse phase conditions described in Figure 3. The (+)-S-enantiomer eluted first fol-

160 ABOUL-ENEIN ET AL.

TABLE 1. Effect of (A) organic modifiers, (B) temperature, and (C) buffer concentration on the chromatographic parameters of the enantiomeric separation of racemic albuterol sulfate

A. Organic modifiers Mobile phase (v/v) a R S

A, no modifier 6.01 7.26 1.21 0.56 A, 0.13% methanol 4.32 5.23 1.21 0.61 A, 0.13% ethanol 4.14 4.95 1.20 0.49 A, 0.13% 1-propanol 3.93 4.50 1.15 0.36 A, 0.13% 2-propanol 3.93 4.64 1.17 0.48 A, 0.13% acetonitrile 4.42 5.32 1.20 0.58

A = 0.01 M phosphate buffer, pH 7.5, ambient temperature.

B. Temperature Mobile phase Temp. "C k; ki a R S

A A A

Ambient 30 37

6.01 5.75 5.53

7.26 6.69 6.26

1.21 0.56 1.16 0.42 1.13 0.38

A = 0.01 M phosphate buffer, pH 7.5, 0.13% vlv 2-propanol.

C. Phosphate buffer concentration [Phosphate buffer], M a R S

0.005 0.008 0.010 0.011 0.012

6.71 4.19 3.97 3.23 3.47

7.62 4.86 4.64 3.19 4.05

1.14 1.16 1.17 1.18 1.17

0.49 0.42 0.48 0.40 0.43

Mobile phase: phosphate buffer, pH 7.5, 0.13% vlv 2-propanol.

lowed by the (-)-R-enantiomer. The effect of organic modifi- ers on the chromatographic parameters is shown in Table 1A. The addition of organic modifiers to the phosphate buffer did not affect the (a) value sigdicantly, with the exception of 1-propanol and 2-propanol. This reflects the importance of hydrophobic binding sites in protein CSPs, which are inhibited by the addition of these organic modifiers. Since hydrophobic interaction is an essential part of the adsorption equilibrium, any reduction (due to the organic modifier) will give a faster elution time. The effect on the capacity factor (k'), however, will depend on the relative decrease in retention times of the two enantiomers. Often, but not always, (a) will be reduced (as is the case with the addition of 1-propanol and 2-propanol) or remain essentially unchanged (as is the case with methanol, ethanol, and acetonitrile additions). The effect of temperature on the resolution of albuterol enantiomers on the AGP column is shown in Table 1B. It is seen that both a and R, values decrease with the increase in temperature. This behavior is as expected with AGP-CSP, as documented by Hermansson. 2o

The effect of the phosphate buffer concentration (ionic strength) on the resolution of the albuterol enantiomers is 'shown in Table 1C. It will be seen that the phosphate ionic strength does not affect the a values, while it slightly affects the resolution factor R, within the ionic strength applied.

Due to the complexity of the molecular interactions with protein phases such as AGP and the variety of binding site

-c-..

INJECl

n- min 0

STOP 1 P---T- 10

Fig. 4. Enantiomeric separation of racemic albuterol sui!ate. Column: Chirex 3022 (250 x 4 mm id.) mobile phase:hexane: 1.2-dichlorOethane: methan0l:tritluo- roacetic acid (160:93160.67, v/v); detector: W 276 m; sample quantity: 17 moles.

161 DIRECT SEPARATION OF ALBUTEROL ENANTIOMERS

TABLE 2. Effect of different organic acids on the chromatographic parameters for the enantiomeric separation of racemic albuterol sulfate

Mobile phase composition" Hexane: 1,2-dichloroethane: Alcohol : Organic acid k; k: a R.

~~

A 200: 116 :20 (methanol) :0.43 (formic acid) 3.65 4.30 1.18 0.72 B 160 93 :16 (methanol) :0.67 (acetic acid) 3.26 3.92 1.20 0.73 C 200: 116 :20 (methanol) :1.78 g (trichloroacetic acid) 2.42 2.88 1.19 0.78 D 160: 93 :16 (methanol) :0.67 (Mluoracetic acid) 2.99 3.62 1.21 0.91 E 176: 103 :31 (ethanol) :0.67 (trifluoroacetic acid) 2.56 3.35 1.31 0.79

"AU mobile phase components are on a volume basis, except for trichloroacetic acid. Other chromatographic conditions are similar to those described in Figure 4.

Fig. 5. (a) Computer-aided schematic representation of interactions between (-)-R-albuterol enantiomer with Chirex 3022 chiral stationary phase. Bold R and S indicate chirality, asterisk indicates the chiral center in albuterol, and circled atomic symbols indicate atoms between which the distances shown have been measured.

factors that cause retention times to vary with analyte concen- tration, high phosphate buffer concentrations are not recom- mended since these anions saturate the binding sites available on the protein surface, thus decreasing the k' values of both enantiomers.

Baseline separation of albuterol enantiomers was achieved using basic Pirkle urea type CSP of (S)-indoline-2-carboxylic acid and (R)-1-(a-naphthy1)ethylamine (Chirex 3022; Fig. 4). The elution order of the enantiomers under normal phase conditions used for thls phase was similar to the AGP column. The effect of organic acids in the mobile phase on the chro- matographic parameters is summarized in Table 2. The R, value increases with the lower pK, acids (i.e., trichloroacetic and trifluoroacetic acid) in the mobile phases containing meth- anol, whereas the a value remains relatively unchanged. The use of a ternary mobile phase comprising hexane, 1,2-dichlo- roethane and an alcohol, was found to be more effective than the use of hexane and alcohol mobile phases. The addition of 1,2-dichloroethane increases the solubility of the solute mole- cules in the mobile phase and also inhibits the adsorption of the alcohol on the CSP. This permits better solute-CSP interac- tions, resulting in a better enantiomeric resolution. Oi e t al. 21

(b) Computer-aided schematic representation of interactions between (+)-S-al- buterol enantiomer with Chirex 3022 chiral stationary phase. Bold R and S indicate chirality, asterisk indicates the chiral center in albuterol, and circled atomic symbols indicate atoms between which the distances shown have been measured.

indicated that the high dipole character of 1,2-dichloroethane is the basis for the interactions with the amide group of the CSP. The possible sites of stereoselective interaction be- tween albuterol and the Chirex 3022 CSP are shown in Figure 5 using HyperChem for Window (Version 3.0) software pro- gram (Autodesk Inc., Sausalito, CAI. The mechanism($ of chiral recognition apparently involves (a) r-n interaction be- tween the aromatic groups on the CSP (naphthyl ring) and the aromatic group of the enantiomers and (b) dipole-dipole inter- actions and hydrogen bonding between the amide and the urea functionalities of the CSP and the polar groups of albuterol, where the secondary alcoholic (-OH) group on the stereo- genic center plays a major part in differentiating between the two enantiomers. The distance between the secondary alco- holic (-OH) group and the carbonyl oxygen urea functionality of the CSP is 4.92 A in the case of (-)-R-enantiomer (Fig. 5a) as compared to 7.25 A for (+)-S-albuterol (Fig. 5b). Thus the transient diastereoisomeric complex formed between (-)-R-albuterol and the CSP is more stable than the corre- sponding (+)-S-albuterol-CSP complex. This explains the elu- tion order observed in the normal phase mode using Chirex 3022-CSP.

162 ABOUL-ENEIN ET AL.

The role of the ethyl group of (R)-l-(a-naphthyl)ethylamine is apparently to provide additional hydrophobic interaction, while the indoline ring system may act as a b a y rigid group to exert its effect through steric hindrance. All of the above interactions between the groups of the CSP and those of the chiral solute combine to form short-lived diastereoisomeric pairs with different energies and stabilities, thus affecting the separation.

The method described is suitable for determining the enan- tiomeric ratio of albuterol enantiomers in pharmaceutical dos- age forms (tablets and injectable) and human plasma after solid phase extraction using a CI8 Sep-Pak cartridge. No interfer- ences were observed in blank human plasma for both phases used in this study. As was expected, the enantiomeric ratio obtained for the pooled human plasma spiked with injectable racemic albuterol sulfate was 5050. Preliminary results showed that a plasma sample obtained from a healthy subject receiving racemic albuterol orally indicated a reduction in the (--)-W( +)-S-enantiomeric ratio. Further studies are in progress in this laboratory.

ACKNOWLEDGMENTS We thank Dr. Zareen Farooqi for his assistance in designing

the computer-aided schematic representations described in these studies. We also thank Mr. Chris Eubanks of Glaxo Inc. (Research Triangle Park, NC) for supplying us with the al- buterol enantiomers and to the King Faisal Specialist Hospital and Research Centre Administration for their continued sup- port of the Bioanalytical and Drug Development Research Program.

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