On-line solid-phase extraction coupled with high-performance liquid chromatography and tandem mass spectrometry (SPE-HPLC-MS-MS) for quantification of bromazepam in human plasma: an automated method for bioequivalence studies.
A validated method for on-line solid-phase extraction coupled with high-performance liquid chromatography tandem mass spectrometry (SPE-HPLC-MS-MS) is described for the quantification of bromazepam in human plasma. The method involves a dilution of 300 muL of plasma with 100 muL of carbamazepine (2.5 ng/mL), used as internal standard, vortex-mixing, centrifugation, and injection of 100 muL of the supernate. The analytes were ionized using positive electrospray mass spectrometry then detected by multiple reaction monitoring (MRM). The m/z transitions 316-->182 (bromazepam) and 237-->194 (carbamazepine) were used for quantification. The calibration curve was linear from 1 ng/mL (limit of quantification) to 200 ng/mL. The retention times of bromazepam and carbamazepine were 2.6 and 3.2 minutes, respectively. The intraday and interday precisions were 3.43%-15.45% and 5.2%-17%, respectively. The intraday and interday accuracy was 94.00%-103.94%. This new automated method has been successfully applied in a bioequivalence study of 2 tablet formulations of 6 mg bromazepam: Lexotan(R) from Produtos Roche Químicos e Farmacêuticos SA, Rio de Janeiro, Brazil (reference) and test formulation from Laboratórios Biosintética Ltda, São Paulo, Brazil. Because the 90% CI of geometric mean ratios between reference and test were completely included in the 80%-125% interval, the 2 formulations were considered bioequivalent. The comparison of different experimental conditions for establishing a dissolution profile in vitro along with our bioavailability data further allowed us to propose rationally based experimental conditions for a dissolution test of bromazepam tablets, actually lacking a pharmacopeial monograph.
Gonçalves
José Carlos Saraiva
JC
Departamento de Medicamentos, Faculdade de Farmácia, Rio de Janeiro, Brazil. gaucho@pharma.ufrj.br
Monteiro
Tânia Maria
TM
Neves
Claúdia Silvana de Miranda
CS
Gram
Karla Regina da Silva
KR
Volpato
Nádia Maria
NM
Silva
Vivian A
VA
Caminha
Ricardo
R
Gonçalves
Maria do Rocio Bencke
Mdo R
Santos
Fábio Monteiro Dos
FM
Silveira
Gabriel Estolano da
GE
Noël
François
F
D000328:Adult; D019540:Area Under Curve; D001960:Bromazepam / Q000097:blood* / Q000302:isolation & purification / Q000493:pharmacokinetics; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D008297:Male; D008875:Middle Aged; D011786:Quality Control; D012995:Solubility; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013810:Therapeutic Equivalency
D001960:Bromazepam