HPLC梯度洗脱法测定膝关节镜术后罗哌卡因关节腔给药的血浆浓度

HPLC gradient elution method for determination of ropivacaine in plasma of intraarticular administration after knee arthroscopy

目的建立RP-HPLC法测定膝关节镜手术后罗哌卡因关节腔给药的血浆浓度。方法以曲马多作为内标;采用Hypersil ODS-C_(18)色谱柱(200 mm×4.6 mm,5μm);流动相为乙腈与0.02 mol·L^(-1) NaH_2PO_4缓冲液(用磷酸调pH 3.7,含0.015%三乙胺)梯度洗脱,流速1.0 mL·min^(-1),紫外检测波长220 nm,血样处理以碱性条件下用乙酸乙酯提取,氮气吹干重溶进样,进样量为20μL。关节镜术后在关节腔中注射罗哌卡因150 mg,于0.25、0.5、0.75、1、1.5、2、4、12、16、24 h取血测定罗哌卡因浓度。结果在选定的色谱条件下,曲马多与罗哌卡因保留时间分别约为6.5、10.6 min,罗哌卡因血浆浓度在0.025～4.0 mg·L^(-1)内线性关系良好(r=0.999 9),最低检测浓度为0.01mg·L^(-1),日内、日间精密度RSD<4%,提取回收率为87.79%～89.20%。样品在室温放置24 h及低温冷冻条件下(-20℃)贮存1个月均稳定。关节腔给药后ρ_(max)为(1.01±0.47)mg·L^(-1),t_(max)为(0.75±0.47)h,t_(1/2)为(9.26±6.23)h。结论该方法灵敏简便,准确,重现性较好,可用于罗哌卡因的血药浓度测定。罗哌卡因关节腔内给药较硬膜外给药缓慢平稳释放入血,产生峰浓度较低,血药浓度持续时间较长,为比较安全的给药方法 。

AIM To establish a RP-I-IPLC method for the determination of ropivacaine in plasma after intra-articular injection in the knee joint following knee arthroscopy. METHODS The sample was treated by adding tramadol as an internal standard. The HPLC method was conducted on a Hypersil ODS （200 mm×4.6 ram, 5 tan） and UV detector at 220 nm. The gradient elution was used with a flow rate of 1.0 mL·min^- 1. The mobile phase was acetonitrile and 0.02 mol·min^- 1 NaH2P04 buffer solution （ containing 0. 015% triethylamine, adjusted pH to 3.7 with phosphoric acid）. The plasma was extracted with ethyl acetate after alkalization and dried under nitrogen. After resolving, the residue was injected into the sample. Patients received ropivacaine 150 mg in intra-articular injection at the end of surgery, blood was drawn from plasma suppliers in 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 12, 16 and 24 h. The plasma concentrations of ropivacaine were determined by HPLC. RESULTS Using this method, the retention time of tramadol and ropivacaine wasabout 6.5 and 10.6 min, respectively. The linear ranges were 0.025 - 4.0 mg·L^- 1（ r = 0.999 9） ; the limit of detection were 0.01 mg·L^- 1 and the relative standard deviation of intm-day and inter-day was less than 4%. The recovery rate of extraction was between 87.79% -89.20%. The sample was stable beth at the room temperture within 24 h and under - 20℃ for 1 month. The Pmax, t max and t1/2 were （ 1.01± 0.47） mg·L^- 1, （0.75 ± 0.47） h and （9.26 ± 6.23） h after intra-artieular administration of mpivaeaine. CONCLUSION The proposed method is simple, accurate and specific for the determination of ropivacaine in human plasma and its pharmacokinetie study after intra-articular administration. The intra-articular administration of ropivacaine provides slower release into blood, lower peak concentration and longer duration of plasma concentration compared with epidural administration. Therefore mpivacaine can be safely administered by intra-artieular injection.