Pharmaceutica Analytica Acta

Huperzine A is a novel sesquiterpene alkaloid isolated from Huperzia serrata [1,2] and it is also derived from other Huperzia species including H. elmeri, H. carinat and H. aqualupian [3]. Huperzine A (CAS No.: 102518-79-6; Figure 1), chemically [(5R,9R,11E)-5-amino-1-ethyldiene- 5,6,9,10-tetrahydro-7-methyl-5,9-methanocloocta-[B]-pyri-din-2(1H)- one] is a potent, selective and slow reversible Acetylcholinesterase (AChE) inhibitor [4,5], which has a mechanism of action similar to donepezil, rivastigmine and galantamine. Huperzine A readily crosses blood brain barrier and shows high specificity for AChE. By using X-ray crystallography it was confirmed that the complexity of huperzine A with AChE Crystal Structure of AChE complexed with huperzine A confirm the specificity of inhibitor [6]. The outcome of clinical trials showed that huperzine A capsule shows is efficient and safe to treat mild to moderate Alzheimer’s disease and also enhance the memory and learning performance of adolescence students [7]. Huperzine A has been approved in China for the treatment of Alzheimer’s disease (in the form of immediate release tablets and capsules) and vascular dementia without any notable side effects [8-12]. It is being marketed as a dietary supplement in USA [12]. Huperzine A is administered orally at a dose range of 0.2-0.4 mg/day for Alzheimer’s disease and 0.06-0.4 mg/day (intramuscularly) for dementia. Following oral administration of 0.4 mg huperzine A tablets to healthy human volunteers peak plasma concentrations (C_max: 2.59 ng/mL ± 0.37 ng/mL) achieved at around 1 h (T_max). The mean α and β half-life was is 21.13 min ± 7.28 min and 716 min ± 130 min, respectively [13].

Figure 1: Structural representation of ZT-1, Huperzine A and Huperzine B.

ZT-1, N-[2-hydroxy-3-methoxy-5-chlorobenzilidene] huperzine A is a novel AChE inhibitor, which rapidly transformed non-enzymatically into its active metabolite huperzine A. The dual mode of action of ZT-1 as an N-methyl-D-aspartate (NMDA) receptor antagonist and an AChE inhibitor, positions it as a third generation anti-Alzheimer’s product by improving the general condition and cognitive functions of affected patients as well as having the potential of being a neuroprotectant [14].

Scope

The aim of this review is to provide: (a) A brief compilation of various analytical methodologies on HPLC, LC-MS and LC-MS/MS for the analysis of huperzine A and ZT-1 in plasma and other biological fluids, which we have captured in a tabular format. It contains sample processing, chromatographic conditions, validation parameters and applicable conclusions; (b) Discussed details about sample preparation, internal standard selection, matrix effect, chromatography and various detection systems. Tables 1 and 2 lists the complete information about the published methods on HPLC, LC-MS and LC-MS/MS on Huperzine A and ZT-1.

Table 1: Summary validation of various published LC-MS/MS methods for ZT-1 and huperzine A. ACN: Acetonitrile; CHCl₃: Chloroform; CSF: Cerebrospinal Fluid; CS-LCMS: Column Switching Liquid Chromatography Mass Spectrometry; DCM: Dichloromethane; ESI: Electro Spray Ionization; F/T: Freeze Thaw; HCl: Hydrochloric Acid; HQC: High Quality Control; HCOONH₄: Ammonium Formate; HILIC: Hydrophilic Interaction Chromatography; AF: Ammonium Formate; IPA: Isopropanol; IS: Internal Standard; i.g.: Intragastric; i.v.: Intravenous; LLOQC: Lower Limit of Quality Control; LQC: Low Quality Control; ME: Matrix Effect; MQC: Medium Quality Control; MRM: Multiple Reaction Monitoring; MeOH: Methanol; Na₂HPO₄: Disodium Phosphate; NaOH: Sodium Hydroxide; NH₃: Ammonia; PTFE: Polytetrafluoroethylene; PK: Pharmacokinetic; RE: Relative Error; RPLC: Reverse Phase Liquid Chromatography; RSD: Relative Standard Deviation; SIM: Selected Ion Monitoring; TCA: Trichloroacetic Acid.

Table 2: Summary validation of various published HPLC methods for ZT-1 and huperzine A. CHCl3: Chloroform; HPLC: High Performance Liquid Chromatography; IS:Internal Standard; i.g.: Intragastric; MeOH: Methanol; RE: Relative Error; RSD: Relative Standard Deviation; UV: Ultra Violet; LQC: Low Quality Control; MQC: Medium Quality Control; HQC: High Quality Control; TEA: Triethanolamine.

Bioanalytical related discussion

A bioanalytical method is useful tool for the quantification of the drugs from the biological matrices (plasma, serum and blood). Extraction methods play a crucial role for the estimation of drugs from these matrices. A good bioanalytical method defined as it is free from other interference substances such as salts, proteins and phospholipids except our analyte of interest. A proper bioanalytical method should be free from endogenous substances like salts, proteins and phospholipids etc.

Sample preparation

To eliminate endogenous components from the matrix, a better sample cleaning process is required. To obtain a better sensitivity and reproducibility the biological matrix should be extracted with an organic solvent(s). For the extraction of huperzine A alone or along with ZT-1 from the biological matrix most of the researchers have used ethyl acetate alone [15-17,18-22] or mixture of other solvent(s) like n-hexane:DCM:2-propanol and CHCl₃:IPA combination [19,20,23-25] or ethyl acetate with isopropanol [20]. Apart from these methodologies Pan et al. have adopted on-line column clean-up process to remove the endogenous components present in the biological sample(s), thus providing the interference free samples for analysis for the quantification [16,17]. Li et al. have used single step liquid-liquid extraction of rat blood samples with ethyl acetate and isopropanol mixture (containing internal standard) and vortex mixed, centrifuged, evaporated and reconstituted in mobile phase [20]. In other reported liquid-liquid extraction methods for the estimation of huperzine A in the biological sample the pH was made to basic because of its pKa value of 7.7 before extraction process [15,18,19]. Some researchers have reported making the plasma pH to 9-12 by adding borax-sodium carbonate buffer to get better recovery in biological matrix [23,24]. Cuthbertson et al. adopted the quantification of Huperzine A from the microscale extraction of clubmoss Huperzia squarrosa using liquid extraction by using CHCl₃ with 99.95% recovery. The detection limit was 20 pg by using UHPLCQTOF- MS instrument [21].

Internal standard (IS) selection

Selection of a proper IS a critical part of the bioanalysis. An ideal IS must meet same extraction recovery, ionization response and almost similar molecular weight of the analyte. Stable Isotopically Labeled (SIL) internal standards give accurate results compared to deuterate IS [26]. Yue et al. reported using a (S)-9, 10-difluoro-3-methyl-7- oxo-2,3-dihydro-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid methyl ester as IS for quantification huperzine A in rat plasma on HPLC with fluorescence detector [23]. Mebendazole was used as IS for the quantification huperzine A in dog plasma on HPLC with UV detector [25]. Some researchers Wei et al. [17] and Wang et al. [19] have been using huperzine B (Figure 1) as an IS to avoid difficulties to measure drug levels on LC-MS/MS [16,18] and Li et al. have also used huperzine B as an IS in quantifying ZT-1 and huperzine A from rat blood [20]. Codeine phosphate [15,22], trimetazidine diHCl [16] and pseudoephedrine HCl [18] were other ISs used while quantifying huperzine A from plasma. Tropane alkaloid atropine was used as an IS for the quantification of huperzine A from plant cuttings of H. saquarrosa [21]. Wei et al. [24] did not use any IS for the quantification of ZT-1 and huperzine A from rat/dog plasma.

Matrix effect

It is defined as an alteration or interference in the sensitivity of an analyte of the sample. For low volatile compounds ionization suppression is a common practice, because of this matrix effect will occur and it can be reduced by doing simple dilution [27]. To get a better recovery and free from sample interference three C18 columns were used for column switching process [17]. Samples can be basified with sodium hydroxide to get rid of ion suppression followed by increasing the sensitivity and recovery of analyte [15]. Two chromatographic separation methods (HILIC and RPLC) were optimized for Wei et al. for the separation of huperzine A in human plasma, RPLC method showed significant levels of matrix effect [22].

Chromatography

A variety of columns were used, with most of the methods a temperature ranging from 25-40°C has been used to achieve the good chromatographic separation of huperzine A in human [15,16,18,22] rat [17,23,24] and dog plasma [19,25] and huperzine A with ZT-1 from rat blood [20] and dog/rat plasma [24]. All the methods are reported by LC-MS or LC-MS/MS used by C₁₈ columns for better peak resolution by using either, isocratic [16-19,22-25] or gradient [20,21] or HILIC [22] separation with a flow rate range between 0.2-1.0 mL/min. Pan et al. [17] used an automated column switching technique with three C₁₈ columns (Agilent SB-C₁₈, Zorbax SB-C₁₈ and C₁₈ guard column) to get a better recovery and reduce the interference from the biological matrix. Also the initial flow rate was 0.15 mL/min which gradually increased by 0.5 mL/min followed by an isocratic separation of huperzine A from the plasma [17]. In HPLC methods, reverse-phase mode C₁₈ [24,25] and normal phase mode C₈ [23] columns were used for validation purpose. Generally the column length ranged from 50-250 mm and the external diameter was 2.1-4.6 mm with a particle size ranging from 3.5-5 μm.

Detection

Major detection methods were mass spectrometry [15-20,22] UHPLC-QTOF-MS [21], HPLC with fluorescence detection [23] and ultra violet detection [24,25]. In current scenario, by using a mass detector is most widely used because of its high sensitivity and selectivity for the detection and estimation from the biological matrices. Currently LC-MS/MS is a popular analytical tool for the quantitative bioanalysis of drugs in pharmaceutical industry due to its superior sensitivity and selectivity. Ye et al. [25] reported anion-paring reverse phase method on dog serum by adjusting the pH to 9.5 by adding borax-sodium carbonate buffer (pH 10.8-11.2). These basic conditions are best suitable for ionized compounds. Adding ionic compounds (sodium dodecyl sulfonate) to the mobile phase to increase the creation of ion pairs with charged analyte to improve the thus the sensitivity of the molecule will be improved. Though Huperzine A is having chromophore at different sites majority of the HPLC methods were reported by ultra violet detection [24,25] at a maximum absorption at 313 nm. An excitation wavelength of 310 nm and emission wavelength of 370 nm was reported by Yue et al. by using a fluorescence detector [23].

Fragmentation of huperzine A mainly happened in the amino group. Li et al. [20] have reported a product ion of m/z 226 and [M+Na]+ ions reported by Zou et al. and Wang et al. respectively [15,19]. Upon fragmentation there is a sequential loss of m/z 243, 226 and 210 was reported by Zou et al.; Li et al.; Wang et al.; Li et al.; Cuthbertson et al.; and Wei et al. respectively [15,18-22] and a Selective Ion Monitors (SIM) method was reported for the quantification of huperzine A in rat plasma and cerebrospinal fluid [17]. In MRM mode, almost all the methods are reported quantitation using by a product ion of m/z 226.

The bioanalysis of ZT-1 and huperzine A gives many challenges during method development for bioanalytical scientists. Based on the review of the literature, following perspectives may consider for the development of bioanalytical methods for ZT-1 and huperzine A.

Huperzine A consists of one ionizable carboxylic group and amino groups with pKa value of 7.7; therefore pH adjustment in the mobile phase is very important to ensure adequate elution of compound of interest from the column. To give better sensitivity or ionization it is important to basify the biological samples.

Due to the presence of chromophoric groups in the structure ideally UV detector should be selected. Selectivity considerations need to be carefully evaluated.

Huperzine A is an important alkaloid to treat various indications of neurodegeneration especially Alzheimer’s disease and also used in the treatment of rheumatism, to relax muscles and improve blood circulation. However, several methods are reported that have independently addressed many of the difficulties associated with the separation and detection of huperzine A. All the reported methods by HPLC, the linearity range from 1-250 ng/mL and very sensitivity sensitive methods compared to HPLC by LC-MS or LC-MS/MS ranging from 0.01-500 ng/mL, respectively. These sensitive LC-MS/MS methods are captured a clear elimination phase after post-dosing with a narrow dose range of 0.1-0.5 mg/kg.

The authors have no conflicts of interest relevant to the ideas and/or contents of the manuscript.