Determination of (R,R)-Formoterol Inversion Products in Human Plasma and Urine By Chiral and Achiral-LC/MS/MSZhe-ming Gu1*, Xian-guo Zhao1, Gary Maier2, and Roger S. H. Hsu2

1XenoBiotic Laboratories, Inc., 107 Morgan Lane, Plainsboro, NJ 08536 ; 2 Sepracor Inc., 84 Waterford Drive, Marlborough, MA 01752

(R,R)-formoterol is a highly selective, potent, and long-acting β2-adrenoceptor agonist currently under development for the maintenance treatment of asthma and the prevention of acutebronchospasm in patients with reversible obstructive airways disease. Formoterol contains two chiral centers with fourdiastereoisomers, i.e., (R,R)-, (R,S)-, (S,R)-, and (S,S)-formoterols. The (S,S)-formoterol is 1,000-fold less potent as a β-agonist than (R,R)-formoterol. In the current study, in vivo chiral inversion of (R,R)-formoterol to one or more of the other three stereoisomers was investigated. Four HPLC methods were established for separation of the four formoterol isomers in human plasma and urine samples. The HPLC effluents were monitored by liquid chromatography-tandem mass spectrometry (LC/MS/MS) with the limit of detection (LOD) at 1 or 3 pg/mL.

Extraction Procedures:Extraction Method 1 (for Chiral-HPLC/MS Method 1)

Human plasma or urine (1 mL) and IS-4 (100 µL) were processed using a SPEC PLUSTM C18 cartridge. The desired eluate was evaporated to dryness under N2 and the residue was reconstituted with 2 mL of 0.4% HCOOH, followed by partition with CH2Cl2. The aqueous fraction was evaporated to dryness and the residue was reconstituted with 50µL of CH3OH:CH3CH2OH (1:1).

Extraction Method 2 (for Achiral-HPLC/MS Method 2)

Human plasma (1 mL) and IS-4 (100 µL) were processed using a SPEC PLUSTM C18 cartridge. The desired eluate was evaporated to dryness under N2 and the residue was reconstituted with 200 µL of CH3OH: H2O (3:7).

Extraction Method 3 (for Achiral-HPLC/MS Method 3)

Human plasma (1 mL) and IS-2 (100 µL) were processed using a SPEC PLUSTM C18 cartridge. The desired eluate was evaporated to dryness under N2 and the residue was reconstituted with 200 µL of CH3OH : CH3CN (4:6).

(R,R)-Formoterol: R = H(R,R)-Formoterol-d6: R = D (Internal Standard, IS)

CR2CRNHCH2CH O CH3

CR3

OH

HO

HNHCO

Liquid chromatography:LC System: Waters 2690 Separations Module or

SHIMADZU Liquid Chromatography

HPLC Method 1 (Fractionation of four isomers)

Column: Chiralcel OJ-H, 5 µm, 250x4.6 mm Mobile Phase: A: Hexane; B: Ethanol containing 1%DEA;

C: CH3OHIsocratic: A:B:C (86:10:4)Flow Rate: 0.5 mL/minInjection Vol.: 15 µL

HPLC Method 2 (Achiral, Acidic LC)

Column: Zorbax Eclips XDB-C18, 5 µm, 2.1x50 mmMobile Phase: A: 0.4% HCOOH in H2O; B: CH3OHGradient: A:B=90:10 (0.5 min), 0.05 min to A:B=30:70

(2.45 min), 1 min to A:B=10:90 (0.05 min), 0.45 min to initial

Flow Rate: 0.3 mL/min (4 min), 0.05 min to 0.4 mL/min Injection Vol.: 35 µL

HPLC Method 3 (Achiral, Basic LC)

Column: Phenomenex, Luna, 3 µm, 100x4.6 mm Mobile Phase: A: 0.02%DEA in H2O; B: CH3CNGradient: A:B=92:8 (2 min), 15 min to A:B=75:25 (5 min),

1 min to initialFlow Rate: 0.4 mL/minInjection Vol.: 15 µL

HPLC Method 4 (Chirabiotic-T, Chiral)

Column: Chiralbiiotic-T, 250x4 mm Mobile Phase: A: CH3OH containing 0.045% HCOONH4

and 0.1% HCOOH; B: EthanolIsocratic: A:B (35:65)Flow Rate: 0.6 mL/minInjection Vol.: 15 µL

Mass Spectrometry:

MS System: PE Sciex API 3000 tandem mass spectrometer coupled with Turbo-Ion Spray interface using multiple reaction monitoring (MRM) detection under positive ion mode

Ion Transitions: Formoterol: m/z 345→ m/z 149Formoterol-d6 (IS): m/z 351 → m/z 155

Figure 1. HPLC-UV Chromatogram of four formoterol isomers(HPLC Method 1)

Figure 5. Mass ion chromatograms of a blank, an LOD (1 pg/mL), and a subject plasma sample (HPLC Method 3)

Figure 6. Mass ion chromatograms of a blank, an LOD, and a subject plasmasample (HPLC Method 4)

Dr. Eckhardt Schmidt, Dr. Weizao Luo, Hao Feng, and Mei Huo from XenoBiotic Laboratories, Inc.

Figure 4. Reconstructed mass ion chromatograms of a human plasma and urine collected from a clinical trial (HPLC Method 1)

Introduction

Experimental

Experimental (Cont.)

Conclusions

Acknowledgement

Results and Discussion

100

12.0 13.0 14.0 15.0 16.0 17.00

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LOD (1 pg/mL)

Subject plasma sample

(R,R) & (S,S)-F-d6 (R,S) & (S,R)-F-d6

(R,R)-F

0

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nsity

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(R,S) & (S,R)-F-d6

(R,R) & (S,S)-F

(R,R) & (S,S)-F-d6

(R,S) & (S,R)-F

36 42 48 540

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36 42 48 540

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Blank

LOD (1 pg/mL)

(S,S)-F

(R,R)-F-d6

(R,R)-F

(S,S)-F-d6

(R,R)-F(R,R)-F-d6 (S,S)-F-d6

Base-line separation of the four formoterol isomers was obtained under the normal phase chiral-HPLC conditions (Figure 1). The plasma and urine extracts (Extraction Procedure 1) were fractionated using the established HPLC method. The HPLC effluents were evaporated, reconstituted, and followed by LC/MS/MS analysis with an LOD at 0.5 pg/mL (Figure 2). The ion chromatograms were reconstructed using Microsoft® Excel. All four isomers were detected in the LOD sample at a concentration of 3 pg/mL (Figure 3). No inversion was observed in human plasma or urine collected from clinical trials (Figure 4).

Although the four formoterol isomers eluted at a single peak under acidic HPLC conditions, the two pairs of enantiomers, i.e., (R,R)/(S,S)-formoterol and (R,S)/(S,R)-formoterol, were separated under HPLC basic conditions using a regular C18 column. The HPLC effluents were monitored by LC/MS/MS with LOD at 1 pg/mL. No inversion of (R,R)-formoterol to (R,S)- or (S,R)-formoterol was observed in human plasma samples (Figure 5).

(R,R)-Formoterol and (S,S)-formoterol were separated using a Chirobiotic-T column (HPLC Method 4). The HPLC effluents were monitored by LC/MS/MS with LOD at 1 pg/mL. No inversion of (R,R)-formoterol to (S,S)-formoterol was observed in human plasma samples (Figure 6).

The separation and determination of four formoterol diastereo-isomers can be achieved using HPLC Method 1. For large-scale sample analysis, the determination of possible inversion products can be obtained using a combination of HPLC Methods 3 and 4. The presence or absence of (R,S)- and (S,R)-formoterols can be determined using HPLC Method 3, and the presence or absence of (S,S)-formoterol can be determined by HPLC Method 4.

Three LC/MS methods were established for separation and determination of four formoterol diastereoisomers in human plasma and urine with an LOD at 1 or 3 pg/mL.

No (R,R)-formoterol inversion products, i.e. (R,S)-, (S,R)-, or (S,S)-formoterol, were detected in human plasma or urine collected from clinical trials.

Formoterol (0.5 pg/mL)

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Blank Sample

(R,R)-F (S,R)-F (S,S)-F(R,S)-F

XenoBiotic Laboratories, Inc.

Figure 2. Mass ion chromatograms of a blank and a LOD sample(HPLC Method 2)

Figure 3. Reconstructed mass ion chromatograms of a blank and an LOD plasma sample (HPLC Method 1)

1.50E+04

3.00E+04

Mas

s Int

ensi

ty (C

PS)

1.50E+04

3.00E+04

Formoterol

Formoterol-d6

BlankFormoterol Formoterol-d6

Time, min

0.00E+00 0.00E+00

0.00E+00

1.50E+03

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Mas

s Int

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0.00E+00

1.50E+04

3.00E+04

FormoterolFormoterol-d6

LOD (3 pg/mL)

(R,R)

(R,S)

(S,R)(S,S)

(R,S)

0.00E+00

1.50E+04

3.00E+04

0.00E+00

2.50E+03

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Mas

s Int

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ty (C

PS)

FormoterolFormoterol-d6

Plasma

Formoterol Formoterol-d6

(R,R)-F-d6

(R,S)-F-d6

(S,R)-F-d6

(S,S)-F-d6

(R,R)-F

0.00E+00

3.00E+05

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PS)

0.00E+00

2.50E+05

5.00E+05

FormoterolFomoterol-d6

Urine

(R,R)-F

(R,R)-F-d6

(R,S)-F-d6

(S,R)-F-d6

(S,S)-F-d6

Time, min