Determination of rosiglitazone and metformin in human serum by CE-ESI-MS

A new method for the determination of anti-diabetic drugs metformin and rosiglitazone based on the use of capillary electrophoresis with electrospray mass spectrometry was developed. The proposed method allowed their separation within 11 min by using 50 mM formic acid at +20 kV. Positive electrospray ionization and selected ion monitoring [M+H](+) of metformin (m/z=130) and rosiglitazone (m/z=358) were performed. Several important experimental parameters influencing electrospray ionization of metformin and rosiglitazone were studied. The final composition of sheath liquid was water/methanol/formic acid (50:49.5:0.5, v/v/v), at a flow rate of 2 μL/min. The developed method was applied for the determination of metformin and rosiglitazone simultaneously in human serum after protein precipitation with acetonitrile. The limits of detection of developed method were 4.42 and 2.14 ng/mL for rosiglitazone and for metformin, respectively, which is sufficient for therapeutic serum concentration levels monitoring for both studied drugs.     

Liquid chromatography/electrospray ionization tandem mass spectrometry analysis of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX)

A quantitative liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method was developed for the analysis of the explosive, octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX). In negative ionization mode, HMX forms an acetate adduct ion [M + CH(3)COO](-), m/z 355, in the presence of a small amount of acetic acid in the mobile phase. The ESI collision-induced dissociation (CID) spectrum of m/z 355 was acquired and the transitions m/z 355 --> 147 and m/z 355 --> 174 were chosen for the determination of HMX in samples. Using this quantification technique, the method detection limit was 1.57 microg/L and good linearity was achieved in the range 5-500 microg/L. This method will help to unambiguously analyze environmentally relevant concentrations of HMX.     

Characterization and quantification of soy isoflavone metabolites in serum of renal transplanted patients by high-performance liquid chromatography/electrospray ionization mass spectrometry

During a dietary intervention study on 16 renal transplanted patients, in which 25 g/day of animal proteins were replaced with 25 g of soy proteins, the metabolic profile of soy isoflavones in serum was characterized. This paper describes a reliable and fast liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) method, in negative ion mode, allowing the characterization and simultaneous quantification of several soy isoflavone metabolites. Six metabolites were identified and quantified: daidzein ([M-H](-) at m/z 252.8), dihydrodaidzein (DHD, [M-H](-) at m/z 254.8), equol ([M-H](-) at m/z 240.9), O-desmethylangolensin (O-DMA, [M-H](-) at m/z 256.8), genistein ([M-H](-) at m/z 268.8), and dihydrogenistein (DHG, ([M-H(+)](-) at m/z 270.8). Quantification was assessed using two deuterated internal standards, D(3)-daidzein and D(4)-genistein. This method permitted a limit of quantification (LOQ, S/N = 10) and a limit of detection (LOD, S/N = 3) of 0.05 microM and 0.005 microM for all analytes, except for genistein, where the LOQ and LOD were 0.005 microM and 0.001 microM, respectively. The linearity ranges were from 0.005 to 1.5 microM for genistein, from 0.05 to 1.5 microM for DHG, and from 0.05 to 0.7 microM for the other metabolites. The relative standard deviations (RSDs) were between 0.19% and 13.9% at the LOQ concentration for all metabolites, and between 0.6% and 4.8% at the maximum concentration. On the basis of the results obtained in the dietary intervention study, it was possible to split the patients into five groups characterized by different metabolic pathways.     

Simultaneous quantification of chlorogenic acid and caffeic acid in rat plasma after an intravenous administration of mailuoning injection using liquid chromatography/mass spectrometry

A simple, rapid and sensitive method was developed for the simultaneous quantification of chlorogenic acid (CGA) and caffeic acid (CA) in rat plasma using a high-performance liquid chromatography system coupled to a negative ion electrospray mass spectrometric analysis. The plasma sample preparation was a simple deproteinization by the addition of two volumes of acetonitrile followed by centrifugation. The analytes and internal standard ferulic acid were separated on an Intersil C8-3 column (5 mm; 250 x 2.1 mm) with acetonitrile/0.05% triethylamine solution (70:30, v/v) as mobile phase at a flow rate of 0.2 mL/min with an operating temperature of 30 degrees C. Detection was performed on a quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source operated in selected ion monitoring (SIM) mode. Negative ion ESI was used to form deprotonated molecules at m/z 353 for chlorogenic acid, m/z 179 for caffeic acid, and m/z 193 for the internal standard ferulic acid. Linear detection responses were obtained for CGA concentrations ranging from 0.005 to 2.0 microg/mL and for CA concentrations ranging from 0.010 to 2.0 microg/mL and the lower limits of quantitation (LLOQs) for CGA and CA were 0.005 and 0.01 microg/mL, respectively. The intra- and inter-day precisions (RSD%) were within 9.0% for both analytes. Deviation of the assay accuracies was within +/-10.0% for both analytes. Their average recoveries were greater than 88.0%. Both analytes were proved to be stable during all sample storage, preparation and analytic procedures. The method was successfully applied to the pharmacokinetic study of CGA and CA following an intravenous dose of 5 mL/kg mailuoning injection to rats.     

超高效液相色谱-串联质谱法测定兔血浆中的丝裂霉素C

建立了采用超高效液相色谱-串联质谱测定兔血浆中丝裂霉素C的方法。以兔空白血浆为基质,通过添加标准溶液的方法配制含丝裂霉素C和内标物曲安奈德的样品,选用乙酸乙酯为提取溶剂,液-液萃取法处理血浆样品。采用Hypersil Gold C18分析柱(50 mm×2.1 mm, 1.9 μm),流动相为甲醇-0.1%甲酸水溶液(90:10, v/v),等度洗脱,流速0.2 mL/min,柱温35 ℃,在3 min内实现了快速分离。采用电喷雾正离子(ESI+)模式电离,选择反应监测(SRM)模式检测,以曲安奈德作为内标物进行定量。用于监测的定量离子对分别为丝裂霉素C m/z 335.2→242.2和曲安奈德m/z 435.2→397.3/415.2,用基质匹配标准溶液法进行定量。结果表明: 兔血浆中丝裂霉素C的质量浓度在1～1000 μg/L范围内线性关系良好(r=0.9978,权重系数(weighting): 1/x2);血浆中丝裂霉素C的检出限(信噪比为3)为0.2 μg/L;其平均回收率为85%～ 115%;日内及日间的相对标准偏差(RSDs)均小于15%,满足生物样品检测的要求。该方法可用于兔气管外壁给药后的血浆样品中丝裂霉素C的检测。本方法选择性强、灵敏度高、操作简便快速、重现性好,适用于丝裂霉素C药代动力学等方面的研究。     

Simultaneous determination of honokiol and magnolol in Magnolia officinalis by liquid chromatography with tandem mass spectrometric detection

An optimized high-performance liquid chromatographic method coupled with tandem mass spectrometric detection (LC-MS/MS) was developed for the simultaneous determination of honokiol and magnolol in Magnolia officinalis. Honokiol and magnolol were separated from the extracts using a reversed-phase C(18) column with a mobile phase consisted of acetonitrile and water (75:25, v/v) at a flow-rate of 0.8 mL/min. Selected reaction monitoring (SRM) mode was used for all sample quantification by the precursor-ion/product ion pair m/z 265 --> m/z 224 for honokiol and m/z 265 --> m/z 247 for magnolol. Validation data showed that this method has good linearity (r(2) > 0.995) over the concentration range of 0.0025-0.5 microg/mL for honokiol and magnolol, and both intra- and inter-day variability were acceptable within 15% at the lowest concentrations for this method. This proposed method provides excellent specificity, higher sensitivity and shorter run time than conventional methods and was applied successfully to determine the contents of honokiol and magnolol in M. officinalis.     

A validated liquid chromatographic/tandem mass spectrometric method for the determination of phencyclidine in microliter samples of rat serum

A liquid chromatographic/tandem mass spectrometric method is described for the determination of phencyclidine (PCP) in small volumes of rat serum (e.g. 50 microl). Samples were extracted using a mixed-mode strong cation-exchange column and then separated isocratically using a narrow-bore (2.1 mm i.d.) 3 microm Hypersil phenyl column and a mobile phase consisting of an ammonium formate buffer (pH 2.7) with 60% (v/v) methanol. Detection was accomplished using positive ion electrospray ionization in the multiple reaction monitoring mode. Mass spectra were obtained and peaks were observed at an m/z (% abundance) of 244 (100), 159 (25), and 86 (89). Tandem mass spectra were also obtained from the m/z 244 precursor ion with peaks observed at m/z 159 (100), 86 (96), and 91 (11). Optimum serum PCP sensitivity and precision were obtained at a transition of m/z 244 --> 159. Matrix-associated ion suppression did not significantly affect the accuracy (100-112%) or precision (CV < or =8%) of the assay. The lower limit of quantitation was 1 ng ml(-1) in 50 microl of serum. The method was used to study the serum pharmacokinetics of PCP in rats after an intravenous bolus dose of PCP.     

Simultaneous determination of harpagoside and cinnamic acid in rat plasma by liquid chromatography electrospray ionization mass spectrometry and its application to pharmacokinetic studies

A simple and sensitive method was developed for the simultaneous quantification of harpagoside and cinnamic acid in rat plasma using high-performance liquid chromatography system coupled to a negative ion electrospray mass spectrometric analysis. The plasma sample preparation was a simple deproteinization by the addition of two volumes of acetonitrile. The analytes were separated on an Intersil C8-3 column (2.1 mm i.d.x250 mm, 5 microm) with acetonitrile-5 mm ammonium formate aqueous solution (60:40, v/v) as mobile phase at a flow-rate of 0.2 mL/min. Detection was performed on a quadrupole mass spectrometer equipped with electrospray ionization (ESI) source operated under selected ion monitoring (SIM) mode. [M+HCOO]- at m/z 539 for harpagoside, [M-H]- at m/z 147 for cinnamic acid and [M-H]- at m/z 137 for salylic acid (internal standard) were selected as detecting ions, respectively. The method was validated over the concentration range 7-250 ng/mL for harpagoside and 5-500 ng/mL for cinnamic acid. The lower limits of quantitation for harpagoside and cinnamic acid were 7 and 5 ng/mL, respectively. The intra- and inter-day precisions (RSD%) were within 9.5% and the assay accuracies (RE%) ranged from -5.3 to 3.0% for both analytes. Their average recoveries were greater than 86%. Both analytes were proved to be stable during all sample storage, preparation and analysis procedures. The method was successfully applied to the pharmacokinetic study of harpagoside and cinnamic acid following oral administration of Radix Scrophulariae extract to rats.     

A rapid and sensitive liquid chromatography/positive ion tandem mass spectrometry method for the determination of cimetropium in human plasma by liquid-liquid extraction

We have developed and validated a simple detection system with high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) for determining cimetropium levels in human plasma using scopolamine butyl bromide as an internal standard (I.S.). The acquisition was performed in the multiple reaction monitoring (MRM) mode, by monitoring the transitions: m/z 357.9 > 103.1 for cimetropium and m/z 359.9 > 103.1 for butyl-scopolamine. The method involves a simple single-step liquid-liquid extraction with dichloromethane. The analyte was chromatographed on an YMC C18 reversed-phase chromatographic column by isocratic elution with 10 mM ammonium formate buffer-methanol (19:81, v/v; adjusted to pH 4.0 with formic acid). The results were linear over the studied range (0.2-100 ng ml(-1)), with r2 = 1.0000, and the total analysis time for each run was 2 min. Intra- and interassay precisions were 0.70-8.54% and 1.08-4.85%, respectively, and intra- and interassay accuracies were 97.56-108.23% and 97.48-103.91%, respectively. The lower limit of quantification (LLOQ) was 0.2 ng ml(-1). At this concentration, mean intra- and interassay precisions were 8.54% and 4.85%, respectively, and mean intra- and interassay accuracies were 97.56% and 98.91%, respectively. The mean recovery ranged from 62.71 +/- 4.06 to 64.23 +/- 2.32%. Cimetropium was found to be stable in plasma samples under typical storage and processing conditions. The devised assay was successfully applied to a pharmacokinetic study of cimetropium bromide administered as a single oral dose (150 mg) to healthy volunteers.     

正相液相色谱-串联质谱法分离普萘洛尔对映体

建立正相液相色谱-串联质谱(LC-MS/MS)分离普萘洛尔对映体的方法,并用于盐酸普萘洛尔片对映体含量测定.样品使用甲醇进行简单提取,采用Chiralcel OD-H手性柱,以正己烷-乙醇-氨水(70∶30∶0.4, v/v/v)为流动相,流速为0.4 mL/min.在正离子模式下,通过电喷雾离子化(ESI+),采用多...     

超高效液相色谱-四极杆飞行时间质谱快速检测动物饲料中10种违禁精神药物

建立了测定动物饲料中三唑仑、艾司唑仑、硝西泮、地西泮、异丙嗪、氯氮平、唑吡坦、甲硫哒嗪、氯丙嗪和咪达唑仑10种违禁精神药物的超高效液相色谱-四极杆飞行时间质谱(UPLC-Q-TOF-MS)方法。样品经混合溶剂甲醇-0.1 mol/L HCl(9:1, v/v)振荡提取,再用MCX固相萃取柱净化处理后,采用UPLC-Q-TOF-MS分析检测。10种化合物在5～100 μg/L范围内均呈良好的线性关系,线性相关系数均大于0.99。本方法的硝西泮、唑吡坦和甲硫哒嗪的定量限(LOQ,以信噪比为10计)为8 μg/kg,三唑仑、艾司唑仑、地西泮、异丙嗪、氯丙嗪和咪达唑仑为10 μg/kg,氯氮平为20 μg/kg。3个添加水平(LOQ、2LOQ、4LOQ)的回收率试验结果表明,10种化合物的回收率在60.6%～108.5%范围内,相对标准偏差均小于10%。本方法可用于动物饲料中违禁精神药物的准确测定。     

Development and validation of a sensitive LC-MS/MS method with electrospray ionization for quantitation of pramipexole in human plasma: application to a clinical pharmacokinetic study

A highly sensitive and specific LC-MS/MS method has been developed and validated for the estimation of pramipexole (PPX) with 500 microL human plasma using memantine as an internal standard (IS). The API-4000 was operated under multiple-reaction monitoring mode (MRM) using the electrospray ionization technique. Solid-phase extraction was used to extract PPX and IS from human plasma. The resolution of peaks was achieved with 0.01 m ammonium acetate buffer (pH 4.4):acetonitrile (30:70, v/v) on a Discovery CN column. The total chromatographic run time was 3.0 min and the elution of PPX and IS occurred at approximately 2.32 and 2.52, respectively. The MS/MS ion transitions monitored were 212.10 --> 153.10 for PPX and 180.20 --> 107.30 for IS. The method was proved to be accurate and precise at linearity range of 20-3540 pg/mL with a correlation coefficient (r) of > or =0.999. The intra- and inter-day precision and accuracy values found to be within the assay variability limits as per the FDA guidelines. The developed assay method was applied to a pharmacokinetic study in human volunteers following oral administration of 0.25 mg PPX tablet.     

Rapid screening method for intact glucosinolates in Chinese medicinal herbs by using liquid chromatography coupled with electrospray ionization ion trap mass spectrometry in negative ion mode

An optimized method using liquid chromatography coupled with electrospray ionization ion trap mass spectrometry (LC/ESI-ITMS) in negative ion mode has been developed for screening different structural classes of intact glucosinolates in six Chinese medicinal herbs. The glucosinolates were extracted with hot methanol/water (70:30 v/v) and separation of the individual glucosinolates was achieved using a reversed-phase C18 column with an aqueous ammonium acetate/methanol gradient. Identification of the intact glucosinolates was based on the detection of compounds with a constant neutral loss of 242 Da corresponding to the combined loss of anhydroglucose (162 Da) and sulfur trioxide (80 Da) in collision-induced dissociation. The structures of the identified glucosinolates were confirmed with the use of group-specific product ions at m/z 195, 241, 259, 275 in their corresponding MS/MS product ion spectra. Differentiation of intact glucosinolates was achieved through their respective retention times and molecular masses as well as the characteristic product ions. The limits of detection were at the low nanogram level per injection, based on constant neutral loss scans. Significant variation in the compositions of intact glucosinolates was identified in the cruciferous herbs. This method was applied in the differentiation and quality control of two pairs of easily confused herbs. Copyright (c) 2008 John Wiley & Sons, Ltd.     

超高效液相色谱-串联质谱法测定大鼠血浆中的磷酸西他列汀

建立了大鼠血浆中磷酸西他列汀含量检测的超高效液相色谱-串联质谱(UPLC-MS/MS)分析方法。以大鼠空白血浆为基质,通过添加标准品的方法配制含磷酸西他列汀和内标物氟西汀的样品,选用甲醇为沉淀剂,经离心除去血浆中的蛋白质,上清液用于目标物的检测。采用Thermo Hypersil Gold C18柱(50 mm×2.1 mm, 1.9 μm)为分析柱,Phenomenex Security Guard C18(4 mm×3.0 mm)为预柱,以乙腈和0.05%(v/v)甲酸水溶液为流动相进行梯度洗脱,流速为200 μL/min, 5 min内实现了快速分离。采用电喷雾正离子(ESI+)模式电离,选择反应监测(SRM)模式检测,确定了磷酸西他列汀和氟西汀的监测离子对分别为m/z 408.0→235.0和m/z 310.0→148.0,用基质匹配标准溶液法进行定量。结果表明: 大鼠血浆中磷酸西他列汀的质量浓度在1～1000 μg/L范围内时线性关系良好(r=0.9991),检出限(信噪比为3)为0.2 μg/L;其平均回收率为85%～115%;日内及日间的相对标准偏差(RSDs)均小于15%,满足生物样品检测的要求。将该方法初步用于大鼠静脉注射后的血浆样品中磷酸西他列汀的检测。该方法快速、灵敏度高、操作简便、重现性好,能够用于磷酸西他列汀药代动力学等方面的研究。     

Preparative purification of gentamicin components using high-speed counter-current chromatography coupled with electrospray mass spectrometry

We developed a useful and preparative method based on high-speed counter-current chromatography with mass spectrometry (HSCCC/MS) to purify gentamicin C1a, C2/2a and C1 from standard powder. The analytes were purified on the HSCCC model CCC-1000 (multi-layer coil planet centrifuge) with a volatile two-phase solvent system composed of n-butanol/10% aqueous ammonia solution (50:50, v/v) and detected on an LCMS-2020EV quadrupole mass spectrometer fitted with an electrospray ionization (ESI) source system in positive ionization following scan mode (m/z 100-500). The HSCCC/ESI-MS peaks indicated that gentamicin C1a (m/z 450: [M+H](+)), C2/2a (m/z 464: [M+H](+)) and C1 (m/z 478: [M+H](+)) have the peak resolution values of 1.3 and 1.7 from 30 mg of loaded gentamicin powder. The HSCCC yielded 3.9 mg of gentamicin C1a, 12.6 mg of gentamicin C2/2a and 12.0 mg of gentamicin C1. These purified substances were analyzed by LC/MS with scan positive-mode. Based on the LC/MS chromatograms and spectra of the fractions, analytes were estimated to be over 95% pure. These gentamicin isomers of C1a, C2/2a and C1 were evaluated for their antibacterial activities. The overall results indicate that this approach of HSCCC/MS is a powerful technique for the purification of gentamicin components.     

Determination of didanosine in human serum by on-line solid-phase extraction coupled to high-performance liquid chromatography with electrospray ionization tandem mass spectrometric detection: application to a bioequivalence study

A method based on solid-phase extraction coupled to liquid chromatography with positive ion electrospray ionization and tandem mass spectrometric detection was developed for the determination of didanosine in human serum, using lamivudine as internal standard. The acquisition was performed in the multiple reaction monitoring mode, monitoring the transitions m/z 237 --> 136.7 for didanosine and m/z 230 --> 111.7 for lamivudine. The method was linear over the range studied (10-1500 ng ml(-1)), with r(2) > 0.98, and the run time was 5 min. The intra- and inter-assay precisions were < or =10% and the intra- and inter-assay accuracies were >95%. The absolute recoveries were 99.8% (10 ng ml(-1)), 98.4% (30 ng ml(-1)), 91.5% (700 ng ml(-1)) and 94.7% (1200 ng ml(-1)). The limits of detection and quantitation were 5 and 10 ng ml(-1), respectively. The method was applied to a bioequivalence study, in which 24 healthy adult volunteers (12 men) received single oral doses (200 mg) of reference and test didanosine formulations (buffered powder for oral solutions), in an open, two-way, randomized, crossover protocol. The 90% confidence interval of the individual ratios (test formulation/reference formulation) for C(max) (peak serum concentration) and AUC(0-inf) (area under the serum concentration versus time curve from time zero to infinity) were within the range 80-125%, which supports the conclusion that the two formulations are bioequivalent regarding the rate and extent of didanosine absorption.     

超高效液相色谱-质谱/质谱联用法测定人血浆中的辛伐他汀

建立了超高效液相色谱-质谱/质谱联用法(UPLC-MS/MS)测定人血浆中辛伐他汀的浓度。血浆样品经乙醚-正己烷-异丙醇(体积比为80∶20∶3)提取,以洛伐他汀为内标,采用ACQUITY UPLCTM BEH C18柱(50 mm×2.1 mm,1.7 μm)分离,以乙腈-10 mmol/L乙酸铵水溶液(体积比为85∶15)为流动相,流速为0.25 mL/min,通过电喷雾离子化,采用多反应监测(MRM)方式进行正离子检测。线性范围为0.051～20.4 ng/mL,日内及日间测定的相对标准偏差不高于10%,平均回收率为91.6%。方法灵敏度高,分析速度快,操作简便,适用于辛伐他汀药物动力学和生物等效性研究。     

Enantiomeric separation and quantification of fluoxetine (Prozac) in human plasma by liquid chromatography/tandem mass spectrometry using liquid-liquid extraction in 96-well plate format

Fluoxetine (Prozac) is currently one of the widely prescribed selective serotonin reuptake inhibitors (SSRIs) for the treatment of depression. A high-throughput sample preparation procedure using liquid-liquid extraction (LLE) in a 96-well plate format in conjunction with liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed and validated for quantification of fluoxetine enantiomers in human plasma. After addition of internal standard and ammonium hydroxide, samples were extracted with ethyl acetate. The organic extract was evaporated to dryness and reconstituted in methanol. Where possible, sample transfer and LLE steps were automated using a Tomtec Quadra 96 workstation. Adequate separation of fluoxetine enantiomeric pairs (resolution of 1.17) was achieved on a vancomycin column eluted with methanol containing 0.075% (by weight) ammonium trifluoroacetate. A triple quadrupole mass spectrometer, operated in the multiple reaction monitoring mode at m/z 310-->44 for fluoxetine enantiomeric pairs and m/z 287-->241 for oxazepam (internal standard), was used. Analysis was performed in the positive ion mode using atmospheric pressure chemical ionization (APCI). The standard curve range was 2.0-1000 ng/mL for each fluoxetine enantiomer. The intra- and inter-day precision and accuracy of the quality control (QC) samples were <12.5% (CV) and <13.6% (CV), respectively, for each fluoxetine enantiomer; the correlation coefficient was >0.990. Method ruggedness was demonstrated by the reproducible performance of the assay during a three-day validation period.     

Simultaneous determination of metformin and glipizide in human plasma by liquid chromatography-tandem mass spectrometry

A method has been developed for the simultaneous quantification of metformin (I) and glipizide (II) in human plasma. It is based on high-performance liquid chromatography with electrospray ionization tandem mass (LC-ESI-MS/MS) spectrometric detection in positive ionization mode. Phenformin (III) and gliclazide (IV) were used as internal standards for I and II, respectively. The MS/MS detection was performed in multiple reaction monitoring (MRM) mode. The precursor-product ion combinations of m/z 130 --> 71, 446 --> 321, 206 --> 60 and 324 --> 127 were used to quantify I, II, III and IV, respectively. This method was validated in the concentration ranges of 0.02-4 microg/mL for I and 0.004-0.8 microg/mL for II. It was utilized to support a clinical pharmacokinetic study after single dose oral administration of a combination of I and II.     

Quantification of morusin using LC–MS in rat plasma: Application to a pharmacokinetic study

A sensitive LC–MS method was developed for the quantification of morusin in rat plasma using praeruptorin C as internal standard. After extraction with diethyl ether, post‐treatment samples were chromatographed on a Hypersil C₁₈ column. An isocratic mobile phase consisting of methanol–water (70:30, v /v) was applied at a flow rate of 0.4 mL/min. Detection was performed via electrospray ionization source with positive ion mode using selected ion monitoring mode at m/z 443.1 for morusin and m/z 451.0 for IS. Acceptable linearity (r ² ≥ 0.99) was observed over the concentration range of 1.5–800 ng/mL. This method was successfully applied in the pharmacokinetics study of morusin in rats.