Bioanalytical Method for Pamabrom ( Determined as 8-Bromotheophylline ) and its Application to a Pharmacokinetic Study

Pamabrom is a common over-the-counter mild diuretic that is often used in combination with acetaminophen and other drugs for the treatment of menstrual pain. It consists of a 1:1 ratio mixture of 8-bromotheophylline and 2-amino-2-methyl-1-propanol.

Pamabrom, a common over-the-counter diuretic, is often used in combination with acetaminophen and other drugs for the treatment menstrual pain [2,3].For example, in a recent clinical trial, the efficacy and safety of two oral medications (with different drug combinations): a test medication containing naproxen sodium (220 mg), paracetamol (300 mg) and pamabrom (25 mg); and a reference medication containing paracetamol (500 mg), pyrilamine (15 mg) and pamabrom (25 mg), were evaluated on primary dysmenorrhea in Mexican women.In this trial, it was concluded that both drug combinations were not different in reducing the dysmenorrheic pain [4].

Study samples, Calibration curve and QC samples
The QCs (low, medium, high) and diluted samples were prepared as described for the calibration samples, at concentrations of 60 ng/mL (LQC), 300 ng/mL (MQC), and 600 ng/mL (HQC), respectively.
Although it is common practice to dissolve the dried extract in the mobile phase, the methanol and water mixture was selected because the extract has better solubility in this mixture than in the mobile phase.There were no compatibility problems with the mobile phase.
Pamabrom concentrations were determined with a column (150 x 4.6-mm, 5-μm particle size, Zorbax® SB-C8, Agilent Technologies, Palo Alto, California) equipped with a pre-column (12.5 x 4.6-mm, 5-μm particle size, Zorbax® SB-C8 (Agilent Technologies, Palo Alto, California) and eluted with a mobile phase consisting of: component A (aqueous ammonium acetate (10 mM, pH 5.0 ± 0.1)) and component B (acetonitrile:water mixture (95:5 v/v)) with an A:B ratio of 83:17 v/v.The column temperature was 15°C, the flow rate was kept at 1 mL/minute, the run time was 6.5 minutes, the injection volume was 20 μL and the analyte detection was carried out at a wavelength of 278 nm.

Method validation:
The method was validated according to Mexican and international guidelines regarding bioanalytical method validation [8][9][10].

Preparation of subject samples
The validation included selectivity, carry-over, calibration curve, lower limit of quantification; accuracy, precision; dilution integrity, and stability.

Data analysis:
The calibration curves were analyzed by linear regression of the peak area ratios (analyte/IS) as a function of the analyte concentrations.
The pharmacokinetic (PK) parameters were: C max : maximum plasma drug concentration, T max : time to reach C max , k e : apparent terminal elimination rate constant, t 1/2 : apparent terminal elimination half-life, AUC 0-t : Area under the plasma concentration-time curve from zero (0) hours to the last measurable concentration (t).AUC 0-∞ : Area under the plasma concentration-time curve from zero (0) hours to infinity (∞).
These were estimated using the non-compartmental approach with WinNonlin Phoenix software version 6.4.

Chromatography
Figure 2 shows several representative chromatograms obtained during the validation of the bioanalytical method.
The retention times (mean±SD) for the analyte and the IS were 4.30±0.81and 5.33±1.02minutes, respectively.

Selectivity
The selectivity was evaluated by analyzing blank human plasma samples from six different subjects, blank human (hemolyzed and lipemic) plasma samples, anticoagulants (lithium and sodium heparin), xanthines (theobromine), and other drug substances commonly used as analgesics (acetylsalicylic acid, ibuprofen, diclofenac, paracetamol and naproxen).No interferences were observed in the resulting chromatograms.
All of the correlation coefficients and the determination coefficients were greater than 0.99, which supports the linearity of the method.
Accuracy was expressed as the % relative error (%RE) between the quantified and the nominal value; and the precision was expressed as % coefficient of variation (%CV).
CV: coefficient of variation; RE: relative error, DS diluted sample Table 3: Dilution integrity evaluation The calibration curve consisted of 7 points with concentration levels of 20, 40, 100, 200, 400, 500 and 800 ng/mL.Thus the range of the curve was 20 ng/mL to 800 ng/mL.The lower limit of quantification (LLOQ) was 20 ng/mL and the upper limit of quantification (ULOQ) was 800 ng/mL.The LLOQ was determined as the lowest concentration of the analyte in a sample which can be quantified reliably, with an acceptable accuracy and precision, which is discussed below.

Calibration curve, lower limit of quantification, linearity, and carry-over
The carry-over of the method was evaluated by injecting blank samples after the injection of the ULOQ sample, and no signals were detected at the expected retention times for either the analyte or the IS in the resulting chromatograms.
The linearity of the method was evaluated by analyzing the data obtained from 4 calibration curves.The corresponding slopes, intercepts, as well as the correlation and determination coefficients are shown in Table 1.The intra-day accuracy and precision of the method was evaluated by analyzing 5 replicates of LLOQ and QCs (low, medium, high).

Accuracy, precision, and dilution integrity
In order to evaluate dilution integrity, two additional samples were analyzed using 5 replicates, at an initial concentration of 1000 ng/mL (higher than the ULOQ), which were further diluted (1:2 and 1:5 dilution ratios).
In a similar manner, the inter-day accuracy and precision of the method were evaluated using 3 analytical runs by analyzing 5 replicates of LLOQ and QCs (low, medium, high).
The results for the accuracy and precision of the method are summarized in Table 2 and those for the evaluation of dilution integrity are shown in Table 3.
CV: coefficient of variation; RE: relative error; LLOQ: lower limit of quantification; LQC: low quality control sample, MQC: medium quality control sample, and HQC: high quality control sample The mean %RE results for both intra-and inter-day accuracy for all tested concentration levels were within the acceptance range of ±20% of the nominal value for the LLOQ and within ±15% of the nominal values for the higher concentration levels.In addition, the %CV results for both intra-and inter-day precision for all tested concentration levels were less than 20% for the LLOQ, and less than 15% for the higher concentration levels [8][9][10].Journal of Bioequivalence Studies For the dilution integrity evaluation, the results of this evaluation indicated that the above mentioned criteria for both accuracy and precision were also met.
The use of the HPLC equipment (Agilent 1200 and 1260) was also evaluated, and the results indicated that the accuracy and precision criteria were met in this case as well (data not provided).
The stability of the analyte was evaluated under the following conditions: freeze and thaw (3 cycles); processed sample at room temperature; evaporated sample (dry extract); in autosampler, long term stability (at -75°C ± 5°C) and stock solutions (analyte and IS).Triplicate QCs (low and high) were used for this evaluation.The results are summarized in Table 4.The results indicated that the stability acceptance criteria were met (all mean %RE values for all tested concentration levels were within ±15% of the nominal values) [8][9][10].

Stability
This indicates that the analyte was stable in plasma for 16 weeks at -75°C ± 5°C and during 3 freeze and thaw cycles.In a sample processed at room temperature for 27 hours; in an evaporated sample for 24 hours at room temperature and in an autosampler for 74 hours at room temperature, and in a stock solution for 3 weeks at 5°C ± 3°C.
The method was applied to a pharmacokinetic study, in which a single dose of 25 mg of pamabrom was orally administered in combination with 300 mg of paracetamol and 220 mg of naproxen sodium to Mexican female subjects.

Pharmacokinetic application
As described above, the pharmacokinetics of pamabrom was studied by determining 8-bromotheophylline, the active diuretic ingredient.
Plasma samples were obtained from 12 subjects (aged 19 to 36 years); who participated in a pharmacokinetic study conducted at a clinical unit (IPHARMA, S.A. de C.V. Monterrey, Nuevo León, Mexico).
The study protocol (B19-15) and the informed-consent form were reviewed and approved by an ethics and research committee and authorized by the Federal Commission for Protection against Sanitary Risks (COFEPRIS).
The study was conducted in accordance with the ethical principles of the Declaration of Helsinki and its amendments, as well as the International Conference on Harmonisation for Good Clinical Practice Guideline.
Blood samples were centrifuged at 3000 rpm for 10 minutes at 5°C.The plasma was separated and stored at -65°C ± 15°C until the samples were transported to Biokinetics (the analytical unit) where they were stored at -75°C ± 5°C until the time of analysis.
The mean plasma concentration-time curve of 8-bromothophylline is shown in Figure 3 and its corresponding semi-logarithmic plot in Figure 4.The main pharmacokinetic parameters are summarized in Table 5.The present method allowed us to characterize the pharmacokinetic profile of pamabrom in combination with naproxen sodium and paracetamol.The sensitivity of the method was satisfactory because its LLOQ of 20 ng/mL was 0.54% of the C max (3685.60 ng/ mL) of 8-bromotheophylline, which should not be greater than 5% of the C max to be acceptable [8][9][10].
The pharmacokinetic profile suggested that the plasma concentrations of 8-bromotheophylline decreased in a biexponential or multiexponential manner.In this case, the estimated k e and its derived pharmacokinetic parameters (t 1/2 and AUC 0-∞ ) should be interpreted with caution because the non-compartmental approach is not suitable for determining these parameters under these circumstances.Therefore, further research is needed to clarify this issue.
With this method, it was possible to analyze a total of 158 samples in a single analytical run, including samples from 6 subjects, the system suitability, QCs, and calibration curve samples.The method could be considered convenient for routine application in pharmacokinetic and bioequivalence studies.

Conclusion
A new sensitive bioanalytical method was developed and validated for the determination of pamabrom, quantified as 8-bromotheophylline using HPLC coupled with UV detection.This method was successfully applied to a pharmacokinetic study, in which a single dose of 25 mg of pamabrom was administered orally in combination with 300 mg of paracetamol and 220 mg of naproxen sodium to Mexican female subjects.

Figure 3 :Figure 4 :
Figure 3: Mean plasma concentration-time curve after a single-dose administration of an oral coated tablet, containing, 25 mg of pamabrom in combination with 220 mg of naproxen sodium and 300 mg of paracetamol (trademark: ANALGEN FEM®, Laboratorios Liomont, S.A. de C.V.) in healthy Mexican female subjects (n = 12).Pamabrom was determined as 8-bromotheophylline.Inset: mean (±SE) concentrations over the first 12 hours after administration

Table 1 :
Linearity of the method

Table 2 :
Accuracy and precision of the method