INTRODUCTION:

Bupropion, an aminoketone [(±)-2-(tert-Butylamino)-1-(3-chlorophenyl)propan-1-one], is an atypical, non-tricyclic, antidepressant drug with different pharmacological mode of action (1). Although it selectively binds to dopamine receptor, its effect is seemed to be due to inhibition of norepinephrine reuptake (2, 3). Apart from antidepressant effect, numerous other therapeutic indications including smoking cessation, sexual dysfunction, obesity, attention deficit hyperactivity disorder and seasonal affective disorder have been attributed to bupropion (4 – 8).

In humans, bupropion undergoes rapid metabolization by cytochrome P450 enzyme (CYP2B6) to pharmacologically active main metabolite hydroxybupropion. Ultimately less than 1% of the oral dose excreted unchanged in man (9-13). The bioavailability of oral formulation of bupropion will therefore be very low indicating the requirement of a highly sensitive and specific method for analysis of bupropion and its metabolites in human plasma. Various methods which are less sensitive, less specific or cumbersome, are available for estimation of bupropion (14 – 17). Even in LCMS/MS method, ion suppression effects due to matrix components present in the reconstituted extracts were observed (18).

Estimation of bupropion by LCMS/MS described here is relatively simple, specific and highly sensitive method which has been validated as per the FDA regulations. It can be used for estimation of this drug for pharmacokinetic analysis and other studies.

EXPERIMENTAL:

Materials and Reagents

Bupropion hydrobromide was purchased from Dipharma, Italy whereas hydroxybupropion and fluoxetine, used as an internal standard were from Varda Biotech, Mumbai. HPLC grade methanol and acetonitrile were obtained from Merck, India. All other chemicals of highest purity grade were locally purchased. Millipore (USA) deionized water was used throughout the procedure.

Calibration standards and quality control samples:

Stock solutions (1mg/ml) of bupropion hydrobromide and hydroxybupropion were prepared in 0.1N HCl. Concentration is then corrected using the potency and actual amount weighed. Working solutions (0.097 -13.533 µg/ml for Bupropion and 0.258 – 35.775 µg/ml for Hydroxy bupropion) were prepared by serial dilution of the stock solution by methanol: water (1:1, v/v). Similarly, stock solution (1mg/ml) of IS, fluoxetine, was prepared in methanol and corrected final concentration is obtained using the potency and amount weighed. Working solution (200ng/ml) was then prepared from this stock solution by serial dilution using methanol: water (1:1, v/v) as diluent. All solutions were stored at 2-8 ºC with due protection from light until analysis.

Calibration standards of concentration range from 1.949 to 270.665 ng/ml and for hydroxybupropion, 5.152 to 715.497 ng/ml, were prepared by adding 196 µl of blank plasma to 4 µl of respective working solution. 50 µl of IS was added to each of these analyte spiked plasma (200 µl). After mixing of 200 µl of 0.5 M Na₂CO_3,2500 µl of Tertiary Butyl Methyl Ether (TBME) was added and kept on the vibromax at 2500 rpm for 15 min. After centrifugation at 4500 rpm for 10 min at 2C, 2000 µl of supernatant was then transferred to a vial containing 20 ml of 0.1 M HCl. It was then dried under nitrogen at 40⁰C in a water bath. The residue was redissolved in 500 µl of mobile phase and 10 µl was injected to LCMS/MS for analysis.

Quality control samples, marked as LLOQC, LQC, MQC and HQC respectively, containing 1.960ng /ml, 4.962 ng /ml, 82.703ng /ml and 206.758 ng /ml of bupropion were prepared in blank plasma. Similarly, quality control samples for hydroxybupropion were prepared in the range of 5.181 ng /ml, 13.117 ng /ml, 218,624 ng /ml, and 546.560 ng /ml.

Sample extraction:

Human blood containing K₃ EDTA as an anti-coagulant was centrifuged at 3500 rpm for 15 min at 4⁰C to separate the plasma. 50 µl of working solution (200ng/ml) of IS was added to each tube except the blank sample and vortexed. Bupropion, hydroxybupropion and IS were isolated from the plasma using liquid – liquid extraction technique as mentioned above and 10 µl was injected to LCMS/MS for analysis.

Chromatography:

The drug and its metabolite were separated on a Zorbax SB C8, 100 mm x 4.6 mm column with particle size 3.5 µ(Agilent) using the binary pump [pump A – 0.2% Formic Acid: pump B – Methanol; 35:65] at a flow rate of 1.0 ml/min in Shimadzu UFLC Prominence attached to API 3000 Mass spectrometer (Applied Biosystems, USA) with an ESI interface. The column oven temperature was maintained at 50⁰C and the run time was 3 min. The analytes were detected on mass spectrometer operating in the positive electrospray ionization mode (split ratio 60:40) with multiple reactions monitoring (MRM). The following precursor product ion transition was monitored for MRM transitions: m/z 240.1 184.1 (Bupropion), m/z 256.0 238.0 (Hydroxybupropion) and m/z 310.0 44.0 (Fluoxetine) with a dwell time of 200 msec. The Turbo Ion Spray (TIS) source temperature was maintained at 450⁰C and TIS voltage was set at 5500 V. Data were acquired and processed with Analyst software 1.4.1.

Matrix ion suppression:

Matrix effect was evaluated by post-column analyte infusion. A standard solution containing 82.703ng /ml of Bupropion, 218,624 ng /ml Hydroxybupropion and 200ng/ml Fluoxetine in mobile phase was infused post-column via a ‘T’ connector into the mobile phase flow (1ml/min) at 10 µl/min employing a Harvard infusion pump. 10µl of extracted blank plasma was then injected into the Zorbax SB C8 column using Shimadzu autosampler and LCMS/MS chromatograms were acquired.

Fig 1a. Bupropion

Fig 1 b: Hydroxy Bupropion

Fig 1 c: Fluoxetine

Result and Discussion:

Specificity and Selectivity:

For selectivity analysis, six different lots of plasma were spiked with analytes (bupropion and hydroxybupropion) and internal standard. Interference at the retention times of analytes and IS was evaluated by comparing peak area response with that of blank plasma. Retention times for hydroxybupropion, bupropion and fluoxetine were 1.31 min, 1.24 min and 1.86 min, respectively (Fig.1a, b and c). No interfering peaks were observed in the blank at the retention times corresponding to drug and I.S. indicating that the procedure is specific to bupropion and its metabolite.

Similarly, no significant matrix effect was found (since percent matrix factor is nearing 100) while analyzing the human plasma samples, calibration standards and QC samples (Table 1).

Table 1: Matrix Effect for estimation of bupropion and its metabolite

Sample	Aqueous LQC Analyte (Area count) (n=6)	Post spiked LQC analyte (Area count ) (n=6)	Percentage matrix factor (n=6)
Bupropion	15037	14752	98.10 + 2.87
Hydroxy bupropion	21603.67	21527.83	99.65 + 2.33
Fluoxetine	286404.83	287367	100.34 + 1.30

Linearity of the Calibration Plot:Calibration plots of bupropion and, hydroxybupropion showed that the calibrations are linear in the concentration ranges of 1.949

ng/mL to 206.758 ng/mL for bupropion and 5.152 ng/mL to 546.560 ng/mL for hydroxybupropion. The respective correlation coefficients were >0.9991 and >0.9986.

Precision and Accuracy:

Intra- or inter- day precision and accuracy were determined by replicate analysis of LLOQC, LQC, MQC and HQC samples. Percent coefficient of variation for intraday precision ranged from 7.30 to 10.59 for Bupropion and 7.85 to 11.77 for Hydroxybupropion which are within acceptable limit (Table 2a and b). Similarly, for interday batch precision %CV ranged from 2.73 to 4.16 for Bupropion and 7.84 to 10.84 for Hydroxybupropion were also within accepted limit (£ 20% at LOQQC and £15% for others).

Intra- or inter- day accuracy were in the range of 95.22% to 101.20% for Bupropion and 93.55% to 96.61% for Hydroxybupropion and 92.86% to 100.51% for Bupropion and 94.12% to 97.09 % for Hydroxybupropion, respectively, which are within the accepted limit (£ 20% at LOQQC and £15% for others)

Recovery:

Absolute recovery percentage was determined by comparing the peak area of bupropion and hydroxybupropion obtained by injecting 6 extracted samples of LQC, MQC and HQC with the peak obtained by injection of standard solutions of the same concentration.

Mean percentage recoveries were 63.6 (Bupropion) and 64.4(Hydroxybupropion) (Table 3). Mean average percent CVs were 4.03 (Bupropion) and 2.35 (Hydroxybupropion) which were well within the accepted limits (£ 20%)

	LOQC			LQC (n = 12)			MQC (n = 12)			HQC (n = 12)
	Actual conc (ng/ml)	Estimated conc. (ng/ml)	% CV	Actual conc. (ng/ml)	Estimated conc. (ng/ml)	% CV	Actual conc. (ng/ml)	Estimated conc. (ng/ml)	% CV	Actual conc. (ng/ml)	Estimated conc. (ng/ml)	% CV
Intraday (n = 12)	1.960	1.987	7.30	4.962	4.787	5.99	82.703	79.717	7.45	206.758	196.873	10.59
Interday (n = 18)	1.960	1.970	4.16	4.962	4.685	2.19	82.703	77.851	0.81	206.758	192.006	2.73

Table 2b: Results from determination of the accuracy and precision of analysis of hydroxybupropion in the quality-control samples

	LOQC			LQC (n = 12)			MQC (n = 12)			HQC (n = 12)
	Actual conc. (ng/ml)	Estimated conc. (ng/ml)	% CV	Actual conc. (ng/ml)	Estimated conc. (ng/ml)	% CV	Actual conc. (ng/ml)	Estimated conc. (ng/ml)	% CV	Actual conc. (ng/ml)	Estimated conc. (ng/ml)	% CV
Intraday (n = 12)	5.11	4.847	11.77	13.117	12.056	6.74	218.624	214.134	3.99	546.56	528.042	7.85
Interday (n = 18)	5.11	4.877	10.84	13.117	12.204	6.78	218.624	208.86	4.41	546.56	530.669	7.84

Table 3: Recovery of bupropion and its metabolite from biological matrix

Sample	LQC			MQC			HQC
Sample	Unextracted area (n=6)	Extracted area (n=6)	Mean %recovery	Unextracted area (n=6)	Extracted area (n=6)	Mean % recovery	Unextracted area (n=6)	Extracted area (n=6)	Mean percentage recovery
Bupropion	16470 + 372	11002 +929	66.80	283078 + 6239	180178 + 3543	63.65	699680 + 14634	423342 + 3671	60.51
Hydroybupropion	23333 + 551	14598 + 550	62.56	381408 + 18213	245208+ 12957	64.29	939518 + 18922	622637+ 16173	66.27

Table 4a: Stability of bupropion

Stability check Procedure	LQC Actual conc. (ng/ml)	LQC Avg. conc. Found+ SD (n=3)ng/ml)	HQCActual conc. (ng/ml)	HQCAvg. conc. Found+ SD (n=3)(ng/ml)
Bench Top (5hrs)	4.962	4.887 + 0.119	206.78	193.12 + 4.149
Freeze Thaw (after 3 Cycles at -70⁰C)	4.962	4.841 + 0.055	206.78	205.95 + 7.525
Auto sampler at 5^o C (after 48 hrs)	4.962	4.735 + 0.282	206.78	204.556 + 7.553

Table 4b: Stability of hydroxybupropion

Stability check Procedure	LQCActual conc. (ng/ml)	LQC Avg. conc. Found+ SD (n=3) (ng/ml)	HQCActual conc. (ng/ml)	HQCAvg. conc. Found+ SD (n=3) (ng/ml)
Bench Top (5hrs)	13.117	12.598 + 0.496	546.56	530.988 + 6.174
Freeze Thaw (after 3 Cycles at -70⁰C)	13.117	12.658 + 0.483	546.56	536.286 + 14.808
Auto sampler at 5^o C (after 48 hrs)	13.117	12.437 + 0.749	546.56	530.235 + 10.294

Table 5a: Short term Stock solution Stability of bupropion and hydroxybupropion in biological matrix

Analyte	Average area for Fresh Analyte (n=6)	Average area for Fresh IS (n=6)	Average Area Ratio	Average area for Analyte (n=6) (after 7 hrs of storage)	Average area for IS (n=6) (after 7 hrs of storage)	Average Area Ratio
Bupropion	239887.833	291730.00	0.8224	243344.833	299580.166	0.8124
Hydroxybupropion	328405.500	291730.00	1.1258	329285.00	299580.166	1.0993

Table 5b: Long term Stock solution Stability of bupropion and hydroxybupropion in biological matrix

Analyte

Average area for Fresh Analyte (n=6)

Average area for Fresh IS

(n=6)

Average Area Ratio

Average area for Analyte (n=6) (after 6 days of storage)

Average area for IS (n=6) (after 6 days of storage)

Average Area Ratio

Bupropion

238911.667

292825.167

0.8162

234923.667

277913.833

0.8470

Hydroxy

bupropion

329088.500

292825.167

1.1241

326764.167

277913.833

1.1785

Stability:

Short – Term/bench - top stability

To check whether the sample is stable during analysis, six aliquots of LQC and HQC samples were thawed and kept at room temperature for 5 hours, which has been decided based on the time required for analysis. The samples were then processed and analyzed as mentioned above. No significant differences were noticed when these results were compared with those obtained from the freshly spiked samples indicating that the analytes (Bupropion and hydroxybupropion) were stable at room temperature (Table 4a and b).

Auto sampler stability

The stability of the processed samples in the auto sampler during analysis was determined by using six aliquots of LQC, MQC and HQC samples. The stability of analytes (Bupropion and hydroxybupropion) and IS were assessed for 48 hours, the expected run time for batches of validation samples. The results were then compared with that of freshly spiked samples. No significant difference in the results indicated that the analytes and IS are stable for at least 48 hour in the auto sampler (Table 4a and b).

Freeze – Thaw stability

Analyte stability was determined after three freeze – thaw cycles for six aliquots of each of the LQC and HQC. The samples were stored below – 70⁰C for 24h and then allowed to thaw at room temperature unassisted. After complete thawing, the samples were stored at same temperature for 12h. The freeze – thaw cycle was repeated twice before analyzing the samples. Comparison of the results with the fresh QC samples indicated no differences (Table 4a and b).

Short term stock solution stability

To ensure that analyte and IS are stable in appropriate solution for a short period of time at room temperature, the stability of stock solutions of bupropion and hydroxybupropion and internal standard were evaluated at room temperature for 7 hours. There were no significant changes in stabilities of any of the stock solutions on keeping at room temperature for 7 hours (Table 5a).

Long term stock solution stability

To evaluate the stability of analyte and IS in appropriate solution for a long period of time under storage condition (2 – 8⁰C), the stability of stock solutions of bupropion and hydroxybupropion and internal standard were evaluated at 2 – 8⁰C for 6 days. Table 5(b) indicates that the stock solutions were stable during the storage.

CONCLUSION:

A simple, accurate, precise, sensitive and reproducible LCMS/MS method has been developed and validated for the determination of bupropion and its metabolite hydroxy bupropion. The use of fluoxetine as internal standard for both the analyte and its metabolite is a simple and economic alternative to expensive deuterated standards used earlier . Moreover, the present liquid – liquid extraction method used for sample extraction produces cleaner samples with no matrix effect as compared to the earlier one.

Acknowledgement:

The authors sincerely thank the management of Norwich Clinical Services for providing the opportunity to complete the project.

References:

1. Stahl SM, Pradko BR, Haight JG, Modell CB, Rockett CB, Learned-coughlin S. (2004) A review of the neuropharmacology of bupropion, a dual norepinephrine and dopamine reuptake inhibitor, Prim. Care Companion. J.Clin.Psychiatry 6: 159 – 166

2. Terry P, Katz JL. (1997) Dopaminergic mediation of the discriminative stimulus effects of bupropion. Psychopharmacology (Berl) 134 (2): 201–212.

3. Learned-Coughlin SM, Bergstrom M, Savitcheva I, Ascher J, Schmith VD, Langstrom B. (2003) In vivo activity of bupropion at the human dopamine transporter. Biol Psychiatry 54 (8): 800–805.

4. Lief HI (1996) Bupropion treatment of depression to assist smoking cessation. Am.J.Psychiatry 153: 442

5. Labbate LA, Grimes JB, Hines A, Pollack MH. (1997) Bupropion treatment of serotonin reuptake antidepressant-associated sexual dysfunction. ANN.Clin. Psychiatry 9: 241-245

6. Plodkowski RA, Nguyen Q, Sundaram U, Nguyen L, Chau DL, Jeor SSt..( 2009) Bupropion and naltrexone: a review of their use individually and in combination for the treatment of obesity. Expert Opin Pharmacother. 10: 1069 -1081.

7. Cantwell DP. (1998) ADHD through the life span: the role of bupropion in treatment. J.Clin. Psychiatry 59 suppl 4: 92 -94,

8. Modell JG, Rosenthal NE, Harriet AE, Krishen A, Asgharian A, Foster VJ., Metz A, Rockett CB., Wightman DS. (2005) Seasonal affective disorder and its prevention by anticipatory treatment with bupropion XL. Bio. Psychiatry 58: 658-667.

9. Hesse LM, Venkatakrishnan K, Court MH, Von Moltke LL, Suan SX, Shader RI, Greenblatt DJ. (2000) CYP2B6 mediates the in vitro hydroxylation of bupropion: potential drug interactions with other antidepressants. Drug Metab. Dispos. 28: 1176 - 1183

10. Faucette SR, Howke RL, Lecluyse EL, Shord SS, Yan B, Laethem RM, Lindley CM. (2000) Validation of bupropion hydroxylation as a selective marker of human cytochrome P450 2B6 catalytic activity. Drug Metabol. Dispos. 28: 1222 - 1230

11. Devane CL, Laizzure SC, Stewart JT, Kolts BE, Ryerson EG, Miller RL. (1990) Disposition of Bupropion in Healthy Volunteers and Subjects with Alcoholic Liver Disease J.Clin.Psychopharmacol. 10: 328 – 332

12. Lindsay DeVane C. (1981) Immediate-Release Versus Controlled-Release Formulations: Pharmacokinetics of Newer Antidepressants in Relation to Nausea Eur. J. Clin. Pharmacol. 21: 127 – 135

13. Lai AA, Schroeder DH. (1983) Clinical pharmacokinetics of bupropion: A review. J. Clin. Psychiatry 44: 82-84

14. Rohrig TP, Ray NG. (1992) Tissue distribution of bupropion in a fatal overdose. J.Anal. Toxicol. 16: 343 – 345

15. Butz RF, Schroeder DH, Welch RM, Mehta NB, Phillips AP, Findlay JWA.( 1981) Radioimmunoassay and pharmacokinetic profile of bupropion in the dog. J. Pharm. Exp. Ther. 217: 602 – 610

16. Plkrajac M, Miljkovic B, Missailovic B.( 1991) Mass spectrometric investigation of 2-aminopropiophenones and some of their metabolites. Rapid Commun. Mass Spectrom. 5: 59 – 61,

17. Zhang D, Yuan B, Qiao M, Li F. (2003) HPLC determination and pharmacokinetics of sustained-release bupropion tablets in dogs. J. Pharm. Biomed. Anal. 33, 287 – 293

18. Borges V, Yang E, Dunn J, Henion J. (2004) High throughput liquid chromatography-tandem mass spectrometry determination of bupropion and its metabolites in human, mouse and rat plasma. J. Chromatogr. B 804:, 277 – 287

Received on 06.02.2012 Modified on 25.02.2012

Asian J. Research Chem. 5(3): March 2012; Page 340-344