A validated LC–MS_MS technique for scientific pharmacokinetics and presumptive section II metabolic pathways following oral administration of Andrographis paniculata extract


Requirements of andrographolide, AP1 (purity = 100.00%); 14-deoxy-11, 12-didehydroandrographolide, AP3 (purity = 99.80%); neoandrographolide, AP4 (purity = 99.67%); and 14-deoxyandrographolide, AP6 (purity = 100.00%) have been equipped by Phytolab GmbH & Co.KG (Vestenbergsgreuth, Germany). The interior normal (IS) of digoxin (purity = 96.6%) was bought from Sigma-Aldrich (St. Louis, MO, USA). The examined product, A. paniculata aqueous extract capsules (101.3% of the label quantity of andrographolide, Lot quantity 119010921) was manufactured by Panaosod Co., Ltd. (Chon Buri, Thailand) in accordance with the standard requirements of Good Manufacturing Observe (GMP). The assay contents of capsule composition have been decided utilizing a validated HPLC–DAD method17, and calculated as 20.26, 6.62, 5.28, 3.02 mg/capsule for AP1, AP3, AP4, and AP6, respectively.

For enzymatic digestion, β-glucuronidase sort IX-A (lyophilized powder) from Escherichia coli, glucuronidase exercise of 1,000,000–5,000,000 items/g protein (glucuronidase exercise = 2,354,185 items/g protein, retested date: 04/02/2020), and sulfatase sort H-1 from Helix pomatia, sulfatase exercise ≥ 10,000 items/g strong (sulfatase exercise = 16,134 items/g strong and β-glucuronidase exercise = 353,820 items/g strong, high quality launch date: 25/05/2021) have been obtained from Sigma-Aldrich (St. Louis, MO). For preparation of phosphate and acetate buffers, sodium dihydrogen phosphate (NaH 2 PO 4 ), disodium hydrogen phosphate (Na 2 HPO 4 ), glacial acetic acid (CH 3 COOH), and sodium acetate (CH 3 COONa), have been obtained from Sigma-Aldrich (St. Louis, MO). Acetonitrile and methanol (HPLC grade) have been bought from Merck (Darmstadt, FR, Germany). Milli-Q water (Millipore, Bedford, MA, USA) was used within the LC–MS/MS system all through the evaluation procedures.


Screening and identification of the analyte and metabolite profiling utilizing LC-QTOF/MS evaluation

For qualitative dedication of untargeted compounds, liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) was carried out on an Agilent 6540 QTOF/MS (Agilent Applied sciences, USA) outfitted with an Agilent 1260 infinity liquid chromatography system. The stationary section of the chromatographic separation was carried out on a 150 mm × 4.6 mm, 5.0 µm Phenomenex Luna C18 column (Phenomenex, USA) utilizing a cellular section consisting of 0.1% formic acid in water pH 2.5–2.7 (A) and 100% acetonitrile (B) at a continuing circulate price of 0.5 mL/min with a managed oven temperature at 35 °C, (and 10 µL) of injection quantity. The elution gradient was began with 30percentB, and was elevated to 90percentB inside 20 min, then maintained at 95percentB throughout 25–30 min adopted by a post-run for five min.

Mass spectrometric evaluation was carried out utilizing an ESI unfavourable and constructive modes with various collision energies (10, 20, and 40 V) over a mass vary of m/z 100–1200 Da. The MS circumstances have been as follows: capillary voltage = 3500 V; circulate price of drying fuel (N 2 ) = 10.0 L/min, 350 °C; nebulizer stress = 30 psi, fragmentation voltage = 250 V for unfavourable mode and 100 V for constructive mode. All acquisition information have been analyzed utilizing MassHunter Software program B.07.01 and MassHunter Qualitative Evaluation Software program B.08.00 (Agilent Applied sciences, USA). To help the metabolite identification, the MS chromatograms have been decided by evaluating the fragmentation patterns of metabolites and library searches in Mass Hunter, and Human Metabolome Database (HMDB). For pattern preparation, pooled urine samples at 0–4 and 4–8 h have been ready utilizing the identical extraction course of described within the pharmacokinetic research.

Improvement of analytical and instrument circumstances for quantitative dedication of 4 main diterpenoids utilizing LC-QqQ-MS/MS evaluation

LC and MS situation optimization

Liquid chromatography-tandem mass spectrometry (LC–MS/MS) was carried out utilizing a Nexera X2 LCMS-8060NX triple quadrupole mass spectrometer (Shimadzu, Japan) outfitted with a SIL-40C XR autosampler, CTO-40C column oven, CBM-40lite system controller, FCV-20AH2 switching valve, DGU-403 degasser, and LC-40D XR solvent supply (pump unit). The LC separation was performed on a VertiSep AQS C18 column (100 mm × 3.0 mm, 3.0 µm) utilizing a gradient cellular section. The completely different solvents (together with acetonitrile, methanol, and water, with or with out formic acid) have been examined to optimize pattern elution programs.

Mass spectrometry (MS) and ionization was optimized and operated in A number of Response Monitoring (MRM) mode. MS detection was totally scanned by straight injecting every normal (10.00–100.00 ng/mL) into the MS instrument with out separation on a column. MS optimization was carried out underneath the constructive and unfavourable circumstances of electrospray ionization (ESI) mode. The MRM transitions and the fragmentations of guardian and daughter ions for every analyte, with the corresponding collision vitality, are proven in Desk 1.

Desk 1 MRM transitions and fragmentations of guardian ions and daughter ions ofandrographolide (AP1); 14-deoxy-11, 12-didehydroandrographolide (AP3); neoandrographolide (AP4); 14-deoxyandrographolide (AP6); and digoxin (IS). Full measurement desk

Preparation of inventory requirements and dealing options

The first inventory resolution (1.00 mg/mL) of the 4 normal diterpenoids (AP1, AP3, AP4, and AP6) and an inside normal (digoxin) have been ready after which saved at − 20 °C. Briefly, every normal was precisely weighed and dissolved within the acceptable quantity of methanol (HPLC grade). Working options containing the 4 diterpenoids have been then ready from the inventory options by serial dilution with methanol to acquire the suitable concentrations of those 4 analytes. For the interior normal (IS), an additional dilution of the inventory resolution was ready at a focus of fifty.00 ng/mL.

Preparation of calibration requirements and high quality management (QC) samples

A calibration curve was generated from completely different normal concentrations that have been ready by spiking a small quantity of the working resolution (no more than 5% of the overall quantity) into clean human plasma or urine to acquire twelve remaining concentrations ranging between 0.98 and 1000.00 ng/mL for every analyte. High quality Management (QC) samples have been additionally ready through the use of the identical technique because the calibration requirements; with concentrations of two.50, 500.00, and 900.00 ng/mL for low (LQC), medium (MQC), and excessive (HQC) concentrations, respectively. These spiked plasma samples (calibration requirements and QC samples) have been then equally extracted following the pattern preparation protocol with the addition of fifty.00 ng/mL of IS.

Plasma and urine samples extraction

An aliquot of fifty µL of plasma or urine was positioned in a 1.5 mL of polypropylene centrifuge micro-tube. Protein was faraway from the pattern by including 200 µL of methanol (containing 50.00 ng/mL of IS). The combination was vortexed for 10 min and was then centrifuged at 12,000 rpm, 4 °C for 10 min. The supernatant was filtered by means of a 0.2-µM PVDF membrane (Chrom Tech) and transferred right into a glass insert in a vial earlier than LC‑MS/MS evaluation.

Technique validation

The bioanalytical technique of LC-QqQ-MS/MS evaluation was totally validated for (1) selectivity and specificity, (2) accuracy and precision, (3) linearity, vary and decrease restrict of quantification, (4) restoration, (5) matrix impact, (6) stability, and (7) hemolyzed plasma impact, based on the US FDA Bioanalytical Technique Validation Guideline15 and the Worldwide Convention on Harmonization ICH Guideline M10 on Bioanalytical Technique Validation16.

Key efficiency traits of technique validation

Selectivity and specificity

The selectivity and specificity have been evaluated by evaluating between the retention time of the extracted clean plasma or clean urine and the retention time of every spiked normal or IS within the extracted samples. If any interferences have been noticed, the sign of their peak space must be lower than 20% of the height space of LLOQ, and fewer than 5% of the common peak space of IS in clean samples.

Accuracy and precision

For within-day accuracy and precision, 4 focus ranges at LLOQ, LQC, MQC, and HQC in 5 replicates, have been decided in the identical day. For dedication of inter-day accuracy and precision, the identical concentrations have been extracted and analyzed on separate days (three completely different days) and used for 3 batch runs in 5 replicates. The accuracy was proven as a share, and the acceptance standards was that every focus must be inside ± 15% for QC samples, and inside ± 20% for the LLOQ. The precision of testing was expressed as share of the coefficient of variation (%CV), and the acceptance standards was that every focus must be lower than 15% for QC samples and fewer than 20% for LLOQ.

Linearity, vary and decrease restrict of quantification (LLOQ)

A twelve-point calibration curve was constructed by plotting the peak-area ratio (analyte:IS) towards the focus of the calibration requirements. Dedication of linearity was executed utilizing 3 calibration curves from every day (three separate days). The evaluation requirement of linear regression was decided by the coefficient of dedication (R2 ≥ 0.99). The acceptance standards for accuracy and precision of the calibration curve must be inside 85–115% and ± 15percentCV of the nominal worth, apart from LLOQ, which must be inside 80–120% and ± 20percentCV.

To find out the decrease restrict of quantification (LLOQ), 5 samples at LLOQ have been analyzed for 3 batch runs on separate days. The analytical sign of LLOQ must be at the least 5 instances increased than the sign of the clean plasma or clean urine. The suitable limits of accuracy must be inside 80–120% and %CV ≤ 20.


The restoration of the 4 diterpenoids in plasma and urine was measured at three completely different QC concentrations: LQC, MQC and HQC (n = 5 for every focus) for each pre-spiked normal of QC samples and post-spiked normal of clean plasma or urine. Extraction recoveries have been calculated by evaluating the height space of pre-spiked normal in a clean matrix with the realm of post-spiked normal in an extracted matrix at corresponding concentrations. To satisfy acceptance standards, the restoration must be exact, correct, and repeatable with %CV of restoration lower than 15%.

Matrix impact

Matrix results at three ranges of QC concentrations (LQC, MQC and HQC) have been decided by evaluating the imply peak space of the samples ready by spiking post-extracted samples with the analytes and IS in resolution (with out extraction) at corresponding concentrations. The %CV of the matrix impact must be inside the acceptance standards, which is lower than 15%. The matrix issue (MF) was estimated based mostly on the equation,

$${textual content{MF }} = { }frac{{{textual content{imply }};{textual content{response}};{textual content{ of }};{textual content{analyte}};{textual content{ or}},{textual content{ IS}};{textual content{ in }};{textual content{post-extracted pattern}},}}{{{textual content{imply }};{textual content{response}},{textual content{ of }};{textual content{analyte}},{textual content{ or }};{textual content{IS}};{textual content{ in}};{textual content{ resolution}}}}$$

The IS normalized MF was then calculated as

$${textual content{IS normalized MF }} = { }frac{{textual content{MF of the analyte}}}{{textual content{MF of IS}}}$$

The acceptance worth of IS normalized MF must be inside 0.8–1.2.


The steadiness of plasma and urine samples have been evaluated at three completely different QC focus ranges (LQC, MQC and HQC, n = 3 for every stage); the circumstances have been freeze–thaw stability, long-term stability, short-term stability, and submit preparative stability in an autosampler. The analysis of stability underneath freeze–thaw situation was carried out over 5 cycles of − 80 °C for at the least 12 h of every cycle. Lengthy-term stability was analyzed after storing the samples at − 80 °C. Quick-term stability of every analyte, for as much as 8 h throughout processing on the bench at room temperature, was additionally evaluated. For analysis of post-preparative stability, the extracted samples have been saved at ambient temperature (4 °C) in an autosampler for twenty-four h. The analyte was thought of as a steady pattern when the common share of accuracy of the QC pattern was within the vary of 85–115% and the %CV was not over 15%.

Hemolyzed plasma impact

To guage the impact of hemolysis of purple blood cells on the quantification of analytes, hemolyzed plasma was ready by including hemolyzed complete blood (3% V/V) into clean plasma16,18, which was then vigorously vortexed. Thereafter, the working normal resolution was added to the hemolyzed plasma at LQC and HQC samples (n = 6 replicates). The determinations for the analytes have been in contrast between concentrations of every analyte within the clean plasma with out hemolysis and the measured focus in hemolyzed plasma. The measurement of all analytes was thought of to not be interfered with hemolysis impact when the common of %accuracy was inside a spread of 85–115% and %CV was ≤ 15%.

Quantitative dedication of conjugated metabolites utilizing enzymatic digestion assay

Optimized enzymatic digestion

To optimize the hydrolysis course of for dedication of conjugated metabolites, enzymatic digestion with glucuronidase and sulfatase was carried out underneath completely different incubation circumstances. The hydrolysis response was initiated by including 50 µL of both enzyme right into a 50 µL of plasma or urine pattern utilizing completely different enzyme concentrations (25, 50, 100, 200, and 400 items/mL for sulfatase and 200, 500, 1000, 2000 and 4000 items/mL for β-glucuronidase). Then the combination was incubated at completely different time factors (T = 15, 30 min and 1, 2, 4 h). The response was terminated by including 150 µL of chilly methanol containing inside normal (50.00 ng/mL). After vortex mixing for 10 min, the extracted samples have been centrifuged at 12,000 rpm, 4 °C, for 10 min. Lastly, 150 µL of supernatant was collected by filtering the combination by means of a 0.2-µM PVDF membrane (Chrom Tech), after which transferred to an insert vial for LC–MS/MS evaluation.

The usual calibration curves of every analyte for figuring out conjugated glucuronide and sulfate metabolites have been carried out utilizing the identical optimized technique as plasma and urine incubation. Briefly, working normal options have been added into clean plasma and urine samples after which the method of incubation was carried out earlier than extraction with protein precipitation.

Technique software to pharmacokinetic research

The validated technique was utilized in pharmacokinetic investigation of 4 main diterpenoids in wholesome topics after oral administration of A. paniculata extract.

Institutional evaluate board and knowledgeable consent assertion

The research protocol was permitted by the Institutional Evaluation Board of the Chulabhorn Analysis Institute (approval date: 28/08/2020, IRB quantity: 062/2563) and in addition registered with the Thai Scientific Trials Registry (approval date: 01/02/2021, TCTR20210201005). All scientific procedures have been carried out in compliance with the Worldwide Convention on Harmonization-Good Scientific Observe (GCP) underneath the Declaration of Helsinki. Knowledgeable consent was obtained from all topics concerned within the research.

Examine design and eligibility standards

The scientific pharmacokinetic research was designed as an open-labeled, with a single oral dosing, and performed underneath a fasting situation. 4 wholesome topics have been recruited based on the next inclusion standards: 18—55 years previous with a physique mass index (BMI) of 18–30 kg/m2. Examine members have been recognized as wholesome based mostly on laboratory testing and bodily examinations. The members acquired 3 capsules containing A. paniculata extract (calculated as 60 mg of andrographolide/dose) earlier than breakfast with 240 mL of ingesting water. Blood samples, in EDTA tubes, have been taken after oral administration at 0, 10, 20, 30, 45 min, and 1, 1.5, 2, 4, 6, 8, 10, 12, 24 h submit dose. All blood samples have been then centrifuged at 4000 ± 100 rpm, 4 °C for five min. Plasma was pipetted and saved in cryotubes at − 80 °C till additional evaluation. Urine samples have been collected throughout 0–4, 4–8, 8–12, 12–24, 24–32, 32–40 and 40–48 h after dosing and have been then saved at − 80 °C earlier than pattern extraction.

Statistical evaluation

Pharmacokinetic parameters have been decided by non-compartmental evaluation utilizing PK options software program, model 2.0 (Summit Analysis Providers). The utmost focus of 4 focused compounds (C max ) and the time to succeed in most focus (T max ) have been taken straight from the person plasma focus versus time profiles. The realm underneath the curve from time zero to the final sampling time (AUC 0-t ) was estimated utilizing the linear trapezoidal rule. The obvious quantity of distribution (Vd/F) and the elimination half-life (t 1/2 ) have been decided utilizing non-compartmental evaluation.

Statistical evaluation was carried out utilizing STATA statistical software program (StataCorp, USA). Steady information have been illustrated as imply ± SD, whereas half-life and T max have been expressed as median [IQR]. Graphs of the comparative pharmacokinetics outcomes have been generated utilizing GraphPad Prism 9.3.0 (GraphPad Software program, USA).

Information availability

The datasets generated and/or analyzed throughout the current research usually are not publicly accessible as a result of moral issues and confidentiality agreements however can be found from the corresponding writer on affordable request that wants a consensus from colleagues.