Determination Of Anti-epileptic Drugs In Human Plasma Using HPLC-MS/MS Method

Objective:To establish a HPLC-MS/MS method for simultaneous determination of the therapeutic drug concentration of topiramate（TPM）,levetiracetam（LEV）,carbamazepine（CBZ）,oxcarbazepine（OXC）and other antiepileptic drugs in human plasma,and to monitor the blood drug concentration of epileptic patients,so as to provide a reference for the individualized medication of patients.Methods:1. HPLC-MS/MS method for simultaneous determination of the con-centration of CBZ and VLX.Column:phenomenex Kinetex XB-C₁₈（50mm×3 mm,2.6μm）;mobile phase:methanol（A）-2%formic acid wate-r（B）,Isocratic elution;flowrate:0.4 m L·min^-1;column temperature:40℃;sample volume:5μL.The internal standard was caffeine.The elec-trospray ion source（ESI）was used for positive ion mode monitoring a-nd the multi-reaction monitoring mode（MRM）was used for determinat-ion of the transition of m/z 273.1→194.2,m/z 279.3→261.3 and m/z 195.1→138.1 for CBZ,VLX and IS.Blood concentrations of CBZ and V-LX were determined by the above method in 10 patients with epilepsy and depression after oral administration of CBZ and VLX for 1 week.2.HPLC-MS/MS method for simultaneous determination of the con-centration of TPM,OXC and its metabolite MHD in plasma of epileps-y patients.Column:phenomenex Kinetex XB-C₁₈（2.6μm,50×3 mm）;m-obile phase:methanol（A）-2%formic acid water（B）;gradient elution:0～2 min,30%A→80%A;2～4 min,80%A;4～5 min,80%A→30%A;flo-w rate:0.4·m Lmin^-1;column temperature:40℃;injection volume:5μL;caffeine was used as an internal standard.The electrospray ion source（ESI）was used for positive ion mode monitoring and the multi-reaction monitoring mode（MRM）was used for determination of the transition of m/z 340.4→127.1,m/z 253.2→236.2,m/z 255.1→237.1,m/z 195.1→138.1 for TPM,OXC,MHD and IS.Blood concentrations of TPM,OXC a-nd their metabolites MHD were determined in 20 epileptic patients after oral administration of TPM and OXC tablets for 2 weeks.3.Simultaneous determination of LEV,OXC and its metabolite MH-D in children with epilepsy by HPLC-MS/MS.Column:phenomenex Ki-netex XB-C₁₈（2.6μm,50×3 mm）;mobile phase:methanol（A）-2%for-mic acid water（B）,gradient elution:0～2 min,30%A→80%A;2～4 emperature:40℃;injection volume:5μL.caffeine was used as an inte-rnal standard.The electrospray ion source（ESI）was used for positive i-on mode monitoring and the multi-reaction monitoring mode（MRM）w-as used for determination of the transition of m/z 171.2→154.2,m/z 253.2→236.2,m/z 255.1→237.1,m/z 195.1→138.1 for LEV,OXC,MHD a-n d IS.Blood concentrations of LEV,OXC and their metabolites MHD w ere determined 2 weeks after LEV and OXC were administered orally i n 20 pediatric patients with epilepsy.Results:1.Blood concentrations of 10 patients with epilepsy and depression were measured as follows:CBZ 5.64～11.56μgm L^-1,VLX 131.8～427.6ng·m L^-1.CBZ had good linear relationship in the range of 0.1～20μg_·m L^-1,and the minimum quantitative limit was 0.01μg·m L^-1.VLX had good linear relationship in 10～800 ng·m L^-1,and the minimum quantitati-y and intra-day precision was less than 15%,and the recovery rate wa-s 89.24%～105.7%.2. Blood concentrations of 20 epileptic patients were measured as:TPM 3.42～10.62μg·m L^-1,OXC 1.22～5.96μg·m L^-1,MHD 11.05～44.91μg·m L^-1.TPM,OXC and MHD had good liner relashionship in 1.00～60.00μg·m L^-1,1.00～50.00μg·m L^-1and 1.00～50.00μg·m L^-1,respectively.The quantitive limits were 1.00μg·m L^-1,1.00μg·m L^-1and 1.00μg·m L^-1 for TPM,OXC and MHD.The average recovery was more than 95%,and the RSD of inter-day and intra-day precision was less than 15%.3. Plasma concentrations of LEV,OXC and MHD in 20 pediatric e-pilepsy patients were measures as follows:LEV 12.46～42.48μg·m L^-1,OXC 1.28～7.66μg·m L^-1,MHD 11.30～47.43μg·m L^-1.LEV,OXC and MHD had good liner relashionship in 1.00～60.00μg·m L^-1,1.00～50.00μ-g·m L^-1 and 1.00～50.00μg·m L^-1,respectively.The minimum quantitative limits were 1.00μg·m L^-1,1.00μg·m L^-1 and 1.00μg·m L^-1,respectively.The average recovery was more than 95%,and the RSD of inter-day an-d intra-day precision was less than 15%.Conclusion:Our study established three analytical methods for the simultaneous determination of blood concentration of two or more drugs in patients with epilepsy.The methods are simple,fast,economical,ha-ve good specificity and sensitivity,and are suitable for the determinatio n of large number of clinical samples.