OBJECTIVE

2

To establish a method for simultaneous determination of plasma concentration of lamotrigine（LTG）， levetiracetam（LEV） and perampanel（PER） in children with epilepsy and apply this method in clinical practice.

METHODS

2

Plasma proteins were precipitated with acetonitrile. Using PER-D

5

as internal standard， ultra-high performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS） method was adopted. The determination was performed on ACQUITY UPLC HSS T3 C

18

column with mobile phase consisted of 0.1% formic acid with 5 mmol/L ammonium acetate-acetonitrile （gradient elution） at the flow rate of 0.3 mL/min. The column temperature was 40 ℃， and sample size was 5 μL. The analysis time was 5

min. The electrospray ionization source and multiple reaction monitoring mode were used for positive ion scanning. The ion pairs used for quantitative analysis of LTG， LEV， PER and internal standard were

m

/

z

255.9→144.9，

m

/

z

171.1→126.1，

m

/

z

350.1→219.0 and

m

/

z

354.9→220.2， respectively. The steady-state trough concentrations of the aforementioned drugs in the plasma of 14 pediatric epilepsy patients receiving combination therapy were determined using the same UPLC-MS/MS method as above.

RESULTS

2

The linear ranges of LTG， LEV and PER were 0.15-24 μg/mL （

R

2

＞0.993）， 0.312 5-50 μg/mL （

R

2

＞0.997） and 6.25-1 000 ng/mL （

R

2

＞0.997）， respectively. The lower limits of quantification were 0.15 μg/mL， 0.312 5 μg/mL and 6.25 ng/mL， respectively. RSDs of intraday and interday precision tests of the three drugs were no more than 9.83%， and the accuracies （relative errors） were between －9.33% and 13.72%（

n

＝6 or

n

＝18）； the average extraction recovery rates were 86.4%-97.9%， and the average matrix effects were 86.9%-110.0% （

n

＝6）. The absolute values of the relative errors in the stability tests were all below 15%. The steady-state trough concentrations of LTG， LEV and PER were （5.64±4.03）μg/mL， （10.67±8.78）μg/mL and（450.20±251.27）ng/mL， respectively； the rates of achieving target trough concentrations were 71.4%， 37.5% and 84.6%， respectively.

CONCLUSIONS

2

The established UPLC-MS/MS method is specific， rapid and suitable for the plasma concentration monitoring in epileptic children receiving combination therapy.