Original

Chromatography, Vol.25 No.2 (2004)

Original

Determination of (R )−3−chlorostyrene oxide in bulk chemicals
Using Liquid Chromatography / Atmospheric Pressure
Chemical Ionization Mass Spectrometry
Masato Kazusaki* and Hirofumi Kawabata
Chemical Analysis Research Group, Pharmaceutical Research & Technology Center, Dainippon Pharmaceutical Co., Ltd, 5−51, Ebie 1−chome, Fukushima−ku, Osaka, 553−0001, Japan
Received for review May 31, 2004. Revised manuscript received July 16, 2004. Accepted August 5, 2004.

Abstract
Liquid chromatography/mass spectrometry (LC/MS) operating condition and the sample preparation method were developed and fully validated according to the guidelines issued by Internal Conference on Harmonization (ICH) for the determination of (R )−3−chlorostyrene oxide(RCSO) present in bulk chemicals. RCSO is the raw material of a certain promising medical chemical. The chromatographic separation ofRCSO and bulk chemical was achieved using a mixture of methanol and acetate buffer (3:2) as the mobile phase. Mass spectrometric analysis
was performed by atmospheric pressure chemical ionization (APCI) mode with positive ion detection. Single ion monitoring (SIM) scanmode of m/z 172.1 was used to quantitatively determine RCSO. Analysis by LC/APCI−MS was carried out within 10 min. Limits of detection
and quantitation level of RCSO in bulk chemicals were estimated to be 5 and 10 ng/mg chemical corresponding to 5 ppm and 10 ppm, respectively. Calibration curve of RCSO exhibited the linearity in the RCSO concentration of 10−120 ng/mg chemical (ppm). Accuracy and intermediate precision of this method were 0.4% and 2.8%, respectively. This analytical method as the impurity testing for quality control wassuccessfully applied to bulk chemicals. RCSO was not detected at all in 32 batches of bulk chemicals tentatively manufactured in our laboratory.
Keywords: (R)−3−chlorostyrene oxide, validation, liquid chromatography−atmospheric pressure chemical ionization mass spectrometry,
ICH guideline

1. Introduction
Styrene oxide is the important chiral stating material for thepharmaceutical industry. [1−4] Particularly, (R )−3−chlorostyreneoxide (RCSO) is the common key material for the preparation ofantagonists of central and peripheral dopamine receptors [5], andseveral promising medicinal substances showing antiobesity andFigure 1. Chemical structure of RCSO.

antidiabetic activities [6]. In the manufacture of a certain promisingchemical in our laboratories, RCSO, that would be positive to theganic impurities present in the drug substance should be lower thanmutagenicity test, is used as the raw material. Chemical structure ofthe qualification threshold of 0.05% or 0.15% depending upon

RCSO is provided in Figure 1.

maximum daily dose of the new drug substance. Qualification

Under the Internal Conference on Harmonization (ICH)

threshold is defined by the ratio of amounts of organic impurities

guideline Q3A (R) “Impurities in the Drug Substances” [7], the or-and bulk drug substance (organic impurity / bulk drug substance).

*Tel: 06−6454−8177, Fax: 06−6458−3723
E−mail: masato−kazusaki@dainippon−pharm.w.jp―６１―

Chromatography, Vol.25 No.2 (2004)

2.3. Preparation of the RCSO quality control solutions

Especially, in the case of highly toxic substance, lower qualification threshold than 0.05% or 0.15% can be important. WheneverChemicals spiked with RCSO was dissolved in the mixture of

the content of organic impurities exceed the qualification threshold,acetonitrile and water (1:1). In this study, the concentration of

the safety−study using animal must be carried out to clarify thechemical was adjusted to 1 mg/mL, unless otherwise stated. Con-

margin of safety of impurity. Therefore, it is crucial to establish thecentrations of RCSO spiked onto bulk chemicals were adjusted to 3

quantitative determination method of RCSO contaminated in bulk

−1000 ng/mL, corresponding to 3−1000 ppm of RCSO contami-chemicals. However, it is difficult to determine the RCSO level innated in bulk chemical. These solutions were employed as the qual-bulk chemicals at ppm level by the conventional HPLC method

ity control solution to validate the analytical method according todue to poor absorption of RCSO molecule for UV−Vis detector

the ICH guidelines, Q2A “Text on Validation of Analytical Proce-equipped in the HPLC system. In our knowledge, the determination

dures” [8] and Q2B “Validation of Analytical Procedures: Method-method of RCSO at ppm level has been published nowhere else.

ology” [9]. Calibration curve was drawn by plotting peak area ofThis report describes the validation characteristics of analytical

RCSO in the chromatogram versus the RCSO concentration in-method for determination of RCSO in bulk chemicals and applica-

jected into LC/MS. Accuracy (%) of the method was defined bytion to quality control of bulk chemicals.

the analysis of the quality control solutions prepared at three concentration levels for accuracy spanning the calibration range. Accuracy was evaluated according to the following equation:

2. Experimental
2.1. Materials and reagentsAccuracy =Bulk chemicals were tentatively manufactured in Dainippon( X −C C ) × 100

Pharmaceutical Co., Ltd. (Osaka, Japan). RCSO was obtained from

where X is the determined RCSO concentration and C is the theo-

Mitsubishi Rayon Co., Ltd. (Tokyo, Japan) and used as the starting

retical RCSO concentration in the quality control solution. Preci-

material of the promising chemical. RCSO is clear and colorlesssion of the method was evaluated by analyzing a certain quality

liquid (boiling point: 120.16℃). Ammonium acetate and acetic

control solution (RCSO concentration of 100 ng/mg chemicals)

acid of reagent grade, and acetonitrile, methanol and water ofsuccessively for “repeatability”, and on 6 different days for “inter-HPLC grade were purchased from Wako Pure Chemical Industries,mediate precision”. Precision was expressed as the percentage ofLtd. (Osaka, Japan). Reagents were used without further purifica-

the relative standard deviation (RSD: %) of replicate measure-tion. Ammonium acetate buffer was prepared by dissolving 0.77 gments.

of ammonium acetate in 800 mL of water, adjusting pH of 4.7 using diluted acetic acid, and adding water to make 1000 mL.

2.4. Application to bulk chemicals

2.2. Instrumentation and conditions

acetonitrile and water (1:1) to be 1 mg/mL. This solution and theBulk chemicals of 32 batches were dissolved in the mixture ofThe HPLC system was Hitachi (Tokyo, Japan) D−7000 sys-

standard solution containing RCSO at 100 ng/mL were injected

tem consisted of a Hitachi Model L−7100 pump, auto−sampler L−

into LC/APCI−MS. The levels of RCSO contaminated in bulk

7200, column oven L−7300 equipped with cooling unit, UV−VIS

chemicals were calculated by the comparison of peak areas ofdetector L−7420 and an interface D−7000. Samples were hold atRCSO obtained from these solutions.

10℃ to prevent the vaporization of RCSO from the sample solution. The LC/MS system was HP 1100 system (degasser: G 1322

3. ResultsA, pump: G 1312 A, auto−sampler: G 1367 A and column control-

3.1. Specificity

ler: G 1316 A) equipped with mass detector 1100 Series LC/MSD

To demonstrate the specificity of this method, the solution(Agilent, CA, USA) used in positive ion (PI) selected ion monitor-containing both RCSO (1000 ng/mL) and chemical (1 mg/mL) wasing (SIM) mode at m/z 172.1. The voltages of fragmentor and cap-

injected into conventional HPLC with UV−detector. The separa-illary were set at 90 V and 2200 V, respectively. Neblizer pressuretion between RCSO and chemical is shown in Figure 2. RCSO andwas set at 50 p.s.i., and drying gas at 4.0 mL/min. Drying gas tem-bulk chemicals were completely resolved within 10 minutes withperature was 340℃, and vaporizer temperature 425℃. Injection

resolution of 9.3. By this conventional HPLC method, limit of de-volume was 50 µL. Analyses were carried out on a conventionaltermination of RCSO was about 0.02% (RCSO / chemical). In theODS−column, Hydrosphere C 18 (75 mm × 4.6 mm i.d.) fromanalytical procedure with LC/MS, the main peak (chemical) was

YMC (Kyoto, Japan) with a mobile phase consisted of methanol

diverted to waste prior to entering the mass spectrometer to avoidand ammonium acetate buffer (3:2), unless otherwise specified.

saturation of the MS detector.

The mobile phase was isocratically pumped at 0.5 mL/min.

―６２―Chromatography, Vol.25 No.2 (2004)

Masato Kazusaki and Hirofumi Kawabata

3.3. Limits of detection and quantitation
The limits of detection (LOD) and quantitation (LOQ) wereAbsorbance(AU)defined by measuring six times the signal of RCSO. The amount of← Chemical

RCSO (ng/mg chemical: ppm) injected into LC/APCI−MS andRSD of peak areas is presented in Table 1. The LOD and LOQ
were taken as the concentration levels at which the RSD were lessthan 30% and 10%, respectively. The LOD and LOQ were 5 ppm←RCSOand 10 ppm of the ratio of RCSO in bulk chemical.

Time(min)Table 1. RCSO level injected into LC/APCI−MS and its relative
standard deviation of peak area (n = 6)

Figure 2. Separation of chemical and RCSO. HPLC operating
conditions: column, Hydrosphere C 18 (75 mm inlength and 4.6 mm in inner diameter); mobile phase, amixture of methanol and ammonium acetate buffer (3:2); flow rate, 0.5 mL/min; detection, UV at 219 nm.

RCSO level (ng/mg chemical: ppm) Relative standard deviation (%)
356.1

526.3

106.6

3.2. Conditions for LC/MS
3.4. Linearity

Before interfacing the LC separation to the MS system, flow
injection analyses were conducted using the atmospheric pressure

The linearity between the peak area and the amount of RCSO

chemical ionization (APCI) and the electrospray ionization (ESI)

injected was evaluated in the range of LOQ (10 ng/mg chemical) tosources in both positive and negative ion modes. The results from120 ng/mg chemical. The plot is depicted in Figure 4. The calibra-ESI ion source screening were insubstantial. No signal of RCSO

tion graph passed through the origin (as judged by the 95 % confi-

was detected in either positive or negative ion mode. Detectabledence level) with correlation coefficient of more than 0.999.

ion signals were realized during the APCI ion source screening in
100

both positive and negative ion modes. A strong signal correspond+

Relative abundance(%)

ing to m/z 172.1 ([M + NH4] ) was observed in the mass spectrum(Figure 3). After establishing that the positive ion APCI source wasmost suitable source for the determination of RCSO, other parameters such as fragmentor voltage, capillary voltage, nebulizer pressure, drying gas flow−rate, drying gas temperature, vaporizer temperature were optimized in order to obtain the intense signal of80
604020RCSO. Selected ion monitoring (SIM) scan mode was selected to0determine RCSO.

020406080

100120140

Amount injected into LC/APCI-MS(ng/mg chemical: ppm)

172.1

100

Relative intensity(%)

Figure 4. Calibration curve generated from peak area and theamount of RCSO injected into LC/APCI−MS. Relative
abundance: 100% = 2.05 E 4.

80

3.5. Accuracy

60

Accuracy was verified by analyzing the given quality controlsolutions. Results are summarized in Table 2. Accuracy shown in40

Table 2 is excellent with values ranging between−4.9% and 3.8%
200100for individual values, and 0.4% for total accuracy. Lower and upper limits of estimated recovery average are−6.2% and 7.0%, re120

140160180200220240

260

280

300

spectively, at the 95% confidence level, indicating this method ism/z

accurate enough as the impurity testing for the purpose of determi-Figure 3. Mass spectrum of RCSO obtained by LC/APCI−MS.

nation of RCSO in the bulk chemicals.

―６３―Chromatography, Vol.25 No.2 (2004)

Table 2. Accuracy of the method
RCSO level (ng/mg chemical: ppm)

Individual accuracy (%)1)

Total accuracy (%)2)

10100
120−4.92.43.80.41) n = 3. 2) n = 9.
(A)

3.6. Precision
9000

Precision was evaluated as the RSD of RCSO assayed values
Absorbance(AU)by six replicate analyses of the solution containing both RCSO(100 ng/mL) and chemical (1 mg/mL) by LC/APCI−MS. RCSO
was successively injected into LC/APCI−MS six times. The RSDof RCSO assayed values (reproducibility) is 1.1%, and the upper
95 percent confidence limit was 2.9%. Two analysts, with three

6000

3000

columns, analyzed solutions containing RCSO and chemical on thedifferent days using three mobile phases separately prepared. Inter0−mediate precision on six separate occasions was 2.8%, and upper

6.5

7.0

limit of RSD was estimated to be 4.8% at 95% confidence level.

7.5

8.0

8.5

Time(min)

These results indicate that this analytical method is precise enoughas the impurity testing for the purpose of determination of RCSO in(B)the bulk chemicals.

9000

Absorbance(AU)3.7. Application to bulk chemicals
Detection of RCSO in bulk chemicals manufactured in ourlaboratories was performed with 32 batches. The time span for thechromatographic run was 10 minutes. The result is summarized inTable 3 and typical chromatograms are shown in Figure 5. It turned

6000

3000

out that RCSO was not detected in bulk chemicals tested at all.
0Table 3. Detection of RCSO in bulk chemicals
Batch no.
A 1, A 2
B 1, B 2, B 3
C 1, C 2, C 3, C 4
D 1, D 2, D 3, D 4, D 5, D 6, D 7, D 8, D 9
E 1, E 2, E 3, E 4, E 5, E 6, E 7, E 8, E 9
F 1, F 2, F 3, F 4, F 5

6.5

7.0

7.5

8.0

8.5

Time(min)RCSO level (ng/mg chemical: ppm)

Figure 5. Selected ion chromatograms of the standard solution (A)and the sample solution prepared from batch #F 1 (B).
<5of RCSO. This analytical result indicate that it is unnecessary toconduct the safety study, which is time−consuming and costly, inorder to clarify the toxicity of RCSO on the treated animals.
Acknowledgement

4. Conclusion
Testing method for determination of RCSO in bulk chemicals

We thank Mr. Nishioka and Miss Umeki for many useful dis-was developed. The RCSO and bulk chemical are completely re-cussions on LC/MS. We are also grateful to Miss Shiono for her

solved by liquid chromatography, and RCSO is determined byexperimental help.

mass spectrometer in the APCI mode. Analytical procedure wascompleted within 10 minutes. This method exhibited a good accu-Referencesracy and reproducibility, and low limits of detection (5 ppm) and[1]Fabio, R. D.; Pietra, C.; Thomas, R. J.; Ziviani, L. Bioorg.
Med. Chem. Lett. 1995, 5, 551−554.

quantitation (10 ppm). The described method was successfully ap[2]plied to 32 batches of the bulk chemicals, resulted in no detection―６４―

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