Brahmi (
The human brain is a complex organ that neuroscientists are still attempting to understand. As people live longer, dysfunction of the brain is becoming a predominant issue for the healthcare system. Cognitive decline, particularly in elderly people, often derives from the interaction between age-related changes and age-related diseases and covers a wide spectrum of clinical manifestations, from intact cognition through mild cognitive impairment and dementia. Neural dysfunction of the brain is becoming a predominant issue for the healthcare system as a result of human longevity.
In recent years, the interest in the use of herbal products has grown exponentially, particularly in the western world as well as in developed countries [
Brahmi [
The herb is from a family Scrophulariaceae and is a small creeping herb with numerous branches, small oblong leaves, and light purple or small and white flowers, with four or five petals. It is found in wetlands throughout the Indian subcontinent in damp and marshy or sandy areas near streams in tropical regions. The genus
Compounds responsible for the pharmacological effects of Bacopa include alkaloids, saponins, and sterols. Detailed investigations first reported the isolation of the alkaloid “brahmine” from Bacopa [
The term bacosides refers to dammarane-type triterpenoid glycosides found in extracts of
Triterpenoid glycosides fall into the broader category of “saponins,” as their amphoteric nature allows them to form emulsions in water. Triterpenoids are widely reported actives in plant based medicines and synthetic analogues have been developed for specific pharmacological functions.
Bacosides were first reported by Chatterji et al. in 1963 [
Jujubogenin MW:472.707.
Early methods for quantification of Bacopa saponins involved conversion to ebelin lactones by acidic hydrolysis and then measuring these by UV-spectrophotometry Pal and Sarin (1992) [
It was not until 2004 that Ganzera et al. [
It is generally expected that compendia methods are validated and can be applied directly without requirement for further validation. While this may be workable for uncomplex pharmaceuticals, it is less realistic when applying methods to complex herbal formulas such as those made from
Validation of analytical methods involves examining the uncertainty associated with each component of a methodological procedure as a means to assessing the suitability of a method for its desired purpose.
General procedures and parameters for validation of analytical methods for the measurement of pharmacologically active substances are guided by regulatory guidelines established by the WHO and PIC/S as well as National Pharmaceutical Compendia such as the BP and the USP.
The method separates bacosides by HPLC using an isocratic mobile phase with detection by UV-Vis detector at 205 nm (Figure
Bacopaside II HPLC by BP method at 205 nm.
We used an Agilent 1100 HPLC with a UV-Vis detector, and a reverse phase Phenomenex Synergi 250 mm
A stock solution of the calibrating reference standard Bacopaside II was prepared diluting 5 mg into 5 mL with methanol (1 mg/mL). This was further serial diluted to create 5-point standard curve across a concentration range of approximately 1.0 to 0.01 mg/mL. The contents of 20 capsules of KeenMind were combined to provide a representative sample. Approximately one gram of the powdered extract was diluted in 70% methanol in 50 mLs and sonicated for 30 minutes, followed by centrifugation. The solution was sampled for injection onto HPLC (Tables
Peak purity values for BP bacoside analytes. Purity is indexed from 0 to 1000.
Compound | Peak purity |
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Bacoside A3 | 973.8 |
Bacopaside II | 951.1 |
Bacoside A | 827.9 |
Bacopasaponin C | 977.8 |
Bacopaside I | 787.5 |
Other peaks | >800 |
% RSD of standard replicate injections across the calibration range.
Concentration (mg/mL) | Mean | Standard deviation | % RSD | |
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LOD |
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62 | 6.81 | 12.53 |
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LOQ |
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94.84 | 9.47 | 9.98 |
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454.86 | 11.42 | 2.51 | |
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Working level |
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934.95 | 6.78 | 0.73 |
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4409.71 | 16.03 | 0.36 | |
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9346.45 | 42.18 | 0.45 |
Accuracy results.
Placebo (%) | Placebo (mg) | Bacopaside II |
Bacopaside II |
(%w/w) |
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0 | 0 | 1 | 0.223 | 100.00 |
80 | 16 | 1 | 0.223 | 104.97 |
100 | 20 | 1 | 0.223 | 102.76 |
120 | 24 | 1 | 0.223 | 100.51 |
Extraction efficiency results.
Extraction number | %w/w bacosides | % recovered |
---|---|---|
1 | 9.235 | 97.7 |
2 | 0.213 | 2.30 |
3 | 0.000 (nd) | 0.00 |
According to the BP, when the chromatograms are recorded using, the prescribed conditions the retention time of Bacopaside II is about 36 minutes. According to the BP, the retention times relative to Bacopaside II are as follows: luteolin, about 0.3; Bacoside A3, about 0.9; Bacoside A, about 1.2; Bacopasaponin C, about 1.3; Bacopaside I, about 1.4 (Figure
Peak areas and retention times of replicate injections of standard solution at 0.089 mg/mL.
Replicate injection number | Peak area | Retention time (min) |
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1.00 | 936.26 | 27.882 |
2.00 | 929.30 | 27.809 |
3.00 | 936.67 | 27.886 |
4.00 | 945.46 | 27.875 |
5.00 | 925.90 | 28.009 |
6.00 | 936.10 | 27.935 |
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Mean |
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STDEV |
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%RSD |
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Bacoside peaks (labelled) included in total Bacosides by BP.
We applied validation procedures to the current BP assay for bacosides in KeenMind
Validation parameters examined included specificity, linearity, limit of detection, limit of quantitation, system precision, method precision, extraction efficiency, intermediate precision, and robustness (Tables
Results of method precision (1st operator) and intermediate precision (2nd operator).
Sample | Injection | Bacosides (%w/w) | |
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replicate | replicate | 1st operator | 2nd operator |
1 | 1 | 1.02609 | 0.9489 |
2 | 1.0181 | 0.9501 | |
3 | 1.0053 | 0.9494 | |
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2 | 1 | 1.03001 | 1.047 |
2 | 1.05028 | 0.9872 | |
3 | 1.03453 | 0.97095 | |
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3 | 1 | 1.00957 | 0.97228 |
2 | 1.02717 | 0.97656 | |
3 | 1.05503 | 0.9754 | |
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4 | 1 | 0.99785 | 0.938 |
2 | 0.9765 | 0.97555 | |
3 | 0.9665 | 0.9839 | |
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5 | 1 | 1.09557 | 0.9748 |
2 | 1.03263 | 1.0428 | |
3 | 1.04265 | 1.01844 | |
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6 | 1 | 0.9804 | 1.0184 |
2 | 0.97473 | 0.9899 | |
3 | 0.99676 | 0.99117 | |
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%w/w (mean) |
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STDEV | 0.031 | 0.024 | |
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% RSD |
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“Specificity” determines that the analyte/s are correctly identified and suitably distinct to allow for accurate measurement. Specificity was assessed initially using the peak purity function on HP Chemstation and by examination of peak profiles and symmetry. Because of the structural similarity of bacosides with respect to their chromophore, the peak purity function was unable to differentiate between different overlapping bacoside peaks. It did however indicate that bacoside analytes were not coeluted with compounds of a different structural class. Table
We purchased available reference standards for the major bacosides to confirm the correctness of the BP peak identification guide which ascribes relative retention times for peaks to be calculated as bacosides relative to the retention time of Bacopaside II. According to this guide Bacopaside I is the large peak eluting at 38.5 min whilst the reference standard purchased from Sigma-Aldrich coelutes with the peak at 22.5 min (Figure
Interestingly, this method does not include bacosides eluting after 29.5 min, which are clearly evident in the trace. This effectively diminishes the value for calculated bacosides in KeenMind from about 30% to about 10%. However, clinical trials which have provided supporting evidence for claims for
To assess linearity, a 6-point set of Bacopaside II, standard solutions prepared in the range of LOQ 150% of the nominal concentration were injected onto HPLC in triplicate. The linearity curve was plotted and the
Bacopaside II calibration curve.
The range of concentration over which the assay is valid is determined by confirming the linear correlation of analyte concentration to instrument response; the limit of detection (LOD); limit of quantitation (LOQ); system and method precision; and accuracy/recovery. We found that the range over which precision, accuracy, and linearity met their defined criteria was from 0.0089–0.89 mg/mL.
LOD (limit of detection) is the concentration at which the analyte is detectable, but where interference from background noise occupies at least 30% (signal : noise ratio = 3) of the peak height, rendering measurement too inaccurate to be recorded. LOQ is the concentration at which the background noise occupies up to 10% (signal : noise ratio = 10) of the peak height, allowing for a reasonable estimate of peak area to be measured.
A series of standard (Bacopaside II) solutions with concentrations ranging from 0.01% of the nominal concentration of the active peak to 100% working strength of the impurities was accurately prepared. Triplicate injections of each were performed and the signal-to-noise ratios determined for all samples starting with the least concentrated. The acceptance criteria for LOD is typically S/N of all 3 injections per solution ≥3.0, which for Bacopaside II is a concentration of 0.00445 mg/mL.
For LOQ, a series of standard solutions was accurately prepared from the LOD concentration to 100% of the standard working concentration. Triplicate injections of each of the above solutions were performed and signal-to-noise ratios of all the solutions determined. The acceptance criteria for LOQ is typically S/N of all 3 injections per solution ≥10.0, which for Bacopaside II is 0.0089 mg/mL.
Range can also be limited by recovery of the analyte from the matrix in which it is bound, prior to extraction for analysis. “Recovery,” also termed “accuracy,” is typically assessed by spiking known amounts of the analyte substance into a formulation or carrier matrix and compared to the amount measured in the samples upon analysis. In our validation of the BP method we applied accuracy by both spiked addition and extraction efficiency studies.
For accuracy by spiked addition, Bacopaside II was added at known concentrations to the product placebo. 2.2 mg Bacopaside II reference standard was accurately weighed into a 10 mL volumetric flask and dissolved and diluted to volume with the solvent mix. 1 mL of this solution was accurately transferred to HPLC sample vials containing 16, 20, and 24 mg placebo and sonicated for 15 minutes. The spiked samples were analysed by HPLC and the amount of Bacopaside II calculated. The % recovery of Bacopaside II from theoretical amounts added was determined and used to indicate the impact on matrix binding of the analyte.
The acceptance criteria for accuracy are that the recovery of Bacopaside II is 90.0% to 110.0%, from concentrations ranging from 80 to 120% of nominal stated content of the analyte. Table
In our validation of the BP bacoside assay we also applied a technique coined “extraction efficiency” wherein the test sample is extracted in series up to 5 times and each serial extract is analysed quantitatively. The total analyte from all serial extracts is determined and the respective % yield of each progressive extract calculated. This approach is used by our laboratory for herbal substances where the plant extract matrix cannot be readily replicated. It measures the efficiency of the extraction process of the analytical method.
To assess extraction efficiency, about 1 g of the sample powder was weighed into a 40 mL vial. 30 mL of methanol/water (70/30) was added and sonicated for 15 minutes. The solution was centrifuged and the supernatant transferred to a 50 mL volumetric flask and made up to mark. Another 15 mL of methanol/water (70/30) was added to the 40 mL vial containing the residual pellet, sonicated for 15 minutes and the centrifugation step repeated. The process was repeated once more and aliquots were sampled from each of the three 50 mL volumetric flasks for injection onto HPLC.
A nominal acceptance criteria of >95% were set for recovery from first extract, which is prepared according to the BP bacoside method. Table
System precision is a measure of the uncertainty associated with the instrument operation and is commonly an outcome of sample injection error. For HPLC systems, precision is assessed by measuring the % RSD of 3–6 repeat injections of the same sample, typically a reference standard dilution. For a HPLC in good operating condition acceptance criteria for the retention time are ≤1.0%, and the % RSD of the peak area is ≤10.0%. The average peak area of bacoside peaks is about 1000 mAU which equates to Bacopaside II at 0.089 mg/mL. At this concentration the instrument attained system precision for injection of Bacopaside II of 0.24% RSD for retention time of 27.889 min and 0.73% RSD for an average peak area of 934.95 mAU.
Method precision, also called repeatability, is a measure of the inherent error in sample preparation. A test sample is prepared 6 times and each preparation injected 3 times. The means of repeat injections of each sample are compared and the % RSD measured.
Table
Intermediate precision is the same task performed by a second operator. A comparison of method precision with intermediate precision is an indication of the human error associated with the sample preparation methodology. This is important as a high level of skill may mask a cumbersome or problematic method.
We attained an intermediate precision result of 2.45% RSD which is comparable to that attained by the first operator. In preparation of herbal specimen for analysis, error can occur with the use of the measuring apparatus, through insufficient extraction of analytes from the product matrix as well as variable peak area calculation affected by poor resolution from other peaks absorbing in the same region at the similar retention time.
Robustness assesses the effect of minor changes to HPLC conditions on the analyte measurement. The robustness of the BP bacoside assay was examined at increased (1.1 mL/min) and decreased (0.9 mL/min) HPLC flow rates, at modified mobile phase buffer concentration (0.7, 0.71, and 0.72% Na2SO4), and also at column temperature of 29°C and 31°C compared with a control at flow rate of 1.0 mL/min and 30°C.
The chromatographic separation of bacosides was negatively affected by all system changes applied.
Figures
[Na2SO4] = 0.70%.
[Na2SO4] = 0.71% showing Bacopasaponin C merged with Bacopaside I.
[Na2SO4] = 0.72% showing Bacoside A3 merged with peak 1.
After twelve years of research at Swinburne University, Melbourne, KeenMind suggests that this clinically proven
We found that the BP Bacopa assay is valid for the analysis of Bacopaside II as the validation results have met the acceptance criteria for this molecule (Table
Summary of validation results.
Test | Limits | Conclusions/results | |
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Specificity | No interfering peaks with that of the target. | Complies, however, bacoside peaks are coeluting | |
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Linearity |
The Y-intercept should not be more than ±2%. | 0.38% | |
Linearity |
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Instrument precision | The % RSD of the retention time is ≤1.0% | 0.24% RSD | |
The % RSD of the peak area is ≤10.0% | 0.073% RSD | ||
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Detection limit |
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0.00445 mg/mL | |
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Quantitation limit |
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0.0089 mg/mL | |
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Method precision | The mean result at method working strength is within the specification | Pass (1.02 %w/w) | |
The % RSD is ≤10.0% | 3.0% RSD | ||
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Intermediate precision | The mean result at method working strength is within the specification | Pass (1.02 %w/w) | |
The % RSD is ≤10.0% | 2.5% RSD | ||
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Extraction efficiency | >95% recovery from first extract | 97.7% of total recovered | |
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Accuracy/recovery | At concentrations ranging from 80 to 120% of nominal stated content, the recovery Bacopaside II is 90.0% to 110.0% | Sample | % |
80 | 100.51 | ||
100 | 102.76 | ||
120 | 104.97 | ||
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Range | Precision, accuracy, and linearity must meet their criteria from LOQ% to 150% of the label claim | 0.00445–0.89 mg/mL |
Even very minor changes to HPLC conditions resulted in large shifts in peak shape and resolution. We found that the quality of separation attained was dependent upon the column condition at the time of use. This creates significant problems in stability trial evaluation where testing time-points are often months apart in which time changes to HPLC and HPLC column condition is inevitable. In order to provide reproducible stability results we run a characterised specimen of KeenMind which is stored frozen and adjust the buffer concentration of the HPLC mobile phase until optimal separation of bacosides is achieved. It is necessary to equilibrate the HPLC column in buffer solution for up to 2 hours before use and condition the column after use with blank injections of butanol. Two blank runs use a butanol injection of 20
Standardisation of herbal medicines is fraught with challenges. While claiming pharmacological efficacy and having clinical evidence to support such claims, the actual mechanisms of activity are often not well understood. While we are confident that bacosides are the active constituents of
Bacosides are saponins and as such have “detergent” like properties. They are therefore more susceptible to subtle change in the solid phase condition of HPLC columns. Without a buffered HPLC system, bacosides do not separate well. But this does not mean they cannot be effectively measured. Ganzera et al. [
Ultimately the most accurate measure of bacosides will be achieved by gravimetric isolation using liquid/liquid partitioning and preparative column chromatography. This approach is more of research activity than being routine analytical so it is not suitable for quality control laboratories. The original ebelin lactone methods by UV-spectrophotometry are simple to apply and could be standardised by comparison to gravimetric results.
The method development and validation study reported in this publication was supported by a grant from Soho Flordis International Pty Ltd (SFI). The terms of this arrangement have been reviewed and approved by the Southern Cross University at Lismore in accordance with its policy on objectivity in research. Dilip Ghosh, one of the authors, is an employee of SFI.