Development And Validation of An Eco-Friendly and High-Throughput HPLC Method To Quantify Pyrethroid and Pyriproxyfen in Long-Lasting Insecticide-Treated Nets

Long-lasting insecticide-treated nets (LLINs) are essential to preventing malaria transmission. The LLINs should last for at least three years, even after repeated washings. Currently, tracking insecticides in LLINs is cumbersome, costly, and requires specialized equipment and hazardous solvents. We therefore developed a low-resource, high-throughput method for detecting insecticides in LLINs. In order to extract insecticides from polyethylene, LLIN samples were heated for 45 minutes at 85 o C in 1-propanol-heptane containing dicyclohexylphthalate as an internal standard. Sample size was reduced to ~0.2 g for reduced extraction volume, which is 10% less than what was recommended. We optimized HPLC chromatographic conditions to assess pyrethroid and pyriproxyfen content in polyethylene-based LLINs. The method is capable of quantifying levels ≥ 0.0015% permethrin, 0.00045% alpha-cypermethrin and 0.00025% pyriproxyfen (w/w) in polyethylene, allowing tracking the insecticides before and after LLINs use. A variety of LLINs can be evaluated with this method, including those with 1% pyriproxyfen (pyriproxyfen-LLIN) or 2% permethrin (Olyset ® Net), 1% pyriproxyfen and 2% permethrin (Olyset ® Duo), or 0.55% pyriproxyfen combined with 0.55% alpha-cypermethrin (Royal Gaurd ® ). Analysis of 120 samples (40 nets) per run can be done with high precision and accuracy, reducing labour time and costs whilst reducing the environmental impact of organic solvents.


Introduction
Human deaths due to malaria declined by approximately 50% between 2000 and 2015 1,2 , primarily due to the development, scale-up and universal distribution of long-lasting insecticide-treated nets (LLINs), the preferred form of insecticide-treated nets (ITNs) 1 . Nearly 2.2 billion ITNs have been delivered worldwide since 2004, of which 1.9 billion (86%) were supplied to Sub-Saharan Africa 3 preventing up to 68% of the malaria cases in the region 2 . ITNs reduce malaria transmission by acting as a physical barrier to block mosquito-human contact and killing and repelling mosquitoes by the insecticide 3,4 .
Pyrethroid insecticides such as permethrin and alpha cypermethrin ( Fig. 1) are neurotoxic voltage-gated sodium channel blockers. The World Health Organization (WHO) recommends using pyrethroid in ITNs as they are highly toxic to mosquitoes but not to mammals 3 . They also have a less irritating smell to humans and excellent excito-repellency e cacy, the characteristic responsible for stimulating behavioural avoidance responses of mosquito malaria vectors from the pyrethroid-treated surface and preventing blood-feeding 3,4 . However, since 2016, there have been worrying signs of malaria resurgence in many areas of Sub-Saharan Africa, primarily due to the rapid evolution of pyrethroid resistance in mosquitoes 3 .
With compelling evidence for the impact of pyrethroid resistance on malaria control 5,6 , LLINs that incorporate two insecticides with different modes of action are being introduced 7,8 . Combining two insecticides with varying modes of action can delay the development of resistance and extend the lifetime of both active ingredients 7,9 . The Olyset® Duo (Sumitomo Chemical Co. Ltd.), for instance, is a prototype net that contains the pyrethroid permethrin plus pyriproxyfen (Fig. 1). The latter is a juvenile growth hormone analogue that prevents metamorphosis in larvae and can reduce the fecundity and longevity of adult mosquitoes. Olyset® Duo has been demonstrated to kill pyrethroid-resistant Anopheles gambiae mosquitoes and sterilize surviving blood-fed mosquitoes 7,10,11 . Recently, Disease Control Technologies (USA) has introduced Royal Guard®, a new type of pyrethroid-pyriproxyfen based LLIN that contains a mixture of alpha-cypermethrin and pyriproxyfen incorporated into the mono lament yarn of a polyethylene polymer. Royal Guard® Net was prequali ed by WHO in March 2019 and can improve malaria vector control as indicated by its increased e cacy against An. gambiae sl mosquitoes before and after 20 standardized washes in laboratory and experimental hut studies 12 .
New ITN products must adhere to guidelines from the WHO Prequali cation Team for Vector Control Products (PQT-VC) in relation to insecticide content, wash resistance, storage stability, bio-e cacy, and eld trials 13 . This requires the parallel development of analytical approaches for new product quality control assessment (QCA). Also, given the imminent arrival of new LLINs into the ITN market, the development of 'accessible' methods for quantifying insecticides will be necessary for stakeholders such as procurement agencies and vector control operatives to monitor the quality of the bed nets being used for malaria control operations. Collaborative International Pesticides Analytical Council (CIPAC) methods that utilize chromatographic techniques are available for insecticide quanti cation 14,15 and referenced in WHO testing speci cations for LLINs 13 . For instance, the standard CIPAC protocol for analyzing pyriproxyfen content in LLIN (715/LN/M, CIPAC Handbook O, page 143) is suitable for determining pyriproxyfen content in nets containing pyriproxyfen as the only active ingredient and in mixtures with permethrin 15 . Also, the HPLC method for pyrethroid quanti cation has been developed to provide a universal protocol for detecting and analyzing pyrethroids from both coated and incorporated nets 16 .
However, all available methods rely on a large sample size (~ 2 grams of net mass equivalent to ~ 400 cm 2 ), consume large volumes of organic solvents that require large extraction vessels and use a rotary evaporator for sample concentration (Fig. 2). Contrary to the aims of green chemistry, there are potential adverse effects to the environment resulting from large volume solvent consumption 17 . Furthermore, these methods are labour-intensive, time-consuming and costly, providing barriers to their being implemented in country for routine QCA.
Here we have modi ed the sampling method of LLINs to reduce the sample size of LLIN and the consumption of organic solvent to simplify the extraction and quanti cation procedure for insecticide(s) in LLINs. In addition, we have optimized the chromatographic conditions used in the standard CIPAC protocol for QCA of pyriproxyfen-LLIN 15 to improve the HPLC sensitivity for pyrethroid quanti cation alone or in combination with pyriproxyfen. A range of prototype and commercial LLINs, i.e. Pyriproxyfen-Net (Pyriproxyfen), Olyset® Net (Permethrin), Olyset® Duo (permethrin and pyriproxyfen mixture) and Royal Guard® (alpha-cypermethrin and pyriproxyfen mixture) were used to assess the optimized method for insecticide(s) quanti cation speci city, accuracy, precession, and reproducibility. Results indicate that the new method is suitable for quantifying insecticide(s) content in LLINs containing pyriproxyfen and/or pyrethroid active ingredient. The new method provides high throughput analytical capacity for insecticide(s) quanti cation in LLINs.

Reagents
Technical grade insecticide standards for HPLC analysis were obtained from Sigma Aldrich -permethrin 98.3% purity (57.8% trans-isomer, 40.5% cis-isomer); alpha-cypermethrin, ≥98% purity). HPLC grade acetonitrile (≥99%), water and heptane were obtained from Fisher Chemicals. 1-propanol (≥99%) was obtained from Across Organics. Four types of LLIN were obtained from different suppliers ( Table 1).  acetonitrile and vortexed for one minute at 2500-3000 rpm before decanting into a 1.5 microcentrifuge tube. The sample was ltered through a PTFE 0.2µm lter before transferring 100µl to an HPLC vial for analysis. Standards of concentrations (31.25µg/, 62.5µg/, 125µg/, 250µg/, 500µg/) were prepared for each insecticide present in the nets being analysed. The HPLC method incorporated an isocratic mobile phase of 70% acetonitrile and 30% water, a 1 /min ow rate, 40-minute run time and an analysis wavelength of 226nm. The quantities of permethrin and pyriproxyfen in g/kg are calculated from standard curves produced from the known standard concentrations and corrected against the internal DCP control ss. The nal insecticide content in g/kg was estimated using the following equation: where: I is the insecticide content in g/kg, and x is the insecticide peak area at 226 nm, (for permethrin the cisand transisomer peak areas were combined). a is the slope of the relevant insecticide standard curve. m is the mass of the net sample. C is the internal standard correction factor, calculated by dividing the average peak area of DCP controls by the DCP peak area obtained for the sample. f is the sample dilution factor .

Speci city
To check the method speci city, chromatogram peaks of extraction solutions from Olyset® Duo® and Royal Guard® were compared with that of analytical grade insecticides (permethrin and pyriproxyfen). We con rmed there was no overlap of the insecticide peaks with either the internal control DCP or contamainants peaks co-extracted from polyethylene matrix. The chromatograms produced from these samples were also analyzed for any obvious peak shouldering, tailing or crossover. The insecticide peak retention time was also compared to that of the injected standards, and the percentage retention time was calculated from the following formula: Linearity Linear regression analysis was used to validate the linearity of HPLC for quanti cation of ve working standard solutions of permethrin, alpha-cypermethrin and pyriproxyfen. The standards used ranged from 31.25µg/ -500µg/ as produced during the net analysis. The average peak area, standard deviation, and relative standard deviation (RSD%) were recorded for each insecticide concentration. By injecting 20 µl of insecticide concentrations 31.25, 62.5, 125, 250 and 500 µg/, the response should be linear with R 2 > 0.9. The linearity was obtained by plotting the peak areas (y, mAU) of insecticide versus injected standard concentration (µg/) onto a column and by the value of their correlation coe cients (R 2 ). For each of the three standard curves produced, the slope value is recorded. The average slope (a), standard deviation (σ) and %RSD of these slopes are also reported.
Limit of detection (LoD) and limit of quanti cation (LoQ) LoD and LoQ assays were performed for both insecticides. According to the HPLC conditions described above, a 20 µl of standard curve ranging from 0.007 -250 µg/ was injected in triplicate. The LoD and LoQ were calculated by regression analysis slope (a) obtained from "eq. 1" and the standard deviation (σ) value of the line obtained by analyzing these low-concentration solutions and following equations:

Accuracy and precision
A recovery experiment was conducted to con rm that insecticides content was determined accurately with high precision. The samples subjected to this assessment were untreated nets forti ed with concentrations of permethrin and pyriproxyfen at the speci cation level for each insecticide. Four nets were analyzed per concentration. The results were analyzed, and the following equation was used for the recoveries of the insecticides calculations: Where R: recovery %, C: observed concentration of the insecticide (µg/) and Cs: forti ed concentration (µg/) permethrin.

Heat stability
A comparative assay was performed to assess the stability of the insecticides when heated to 85 o C for 45 minutes, comparing results with and without heating. For the heat stability experiment, 5 of insecticide at two concentrations, 0.4 and 0.2 mg/ (w/v) in extraction solution were heated in triplicate at 85°C for 45 minutes. 1 of the solution was removed, evaporated, and reconstituted in 1 of HPLC-grade acetonitrile for HPLC analysis. In parallel, 1 unheated samples from the insecticide standard were evaporated and reconstituted in 1 acetonitrile to compare HPLC chromatograms of heated versus unheated treatments. All samples were then treated the same as described in the test method. The average insecticide recovered, standard deviation and %RSD for heating and non-heating methods were reported for each insecticide.

Reproducibility test
To assess the method reproducibility, 24 new nets from Olyset® and Olyset® Duo (Table 1) were analyzed in triplicate. For Royal Guard® LLIN, one net was analyzed ten times alongside 30 net analyzed in a single replicate. All net samples were extracted and analyzed following the modi cation described above.

Improvement of HPLC analysis
To increase the HPLC sensitivity for the simultaneous analysis of pyriproxyfen and pyrethroids in LLINs, we optimized the analytical chromatographic conditions in the standard CIPAC protocol recommended for quantifying pyriproxyfen in pyriproxyfen-LLIN 15 . Olyset® Duo LLIN manufactured with 20 g/kg permethrin (2% w/w) and 10 g/kg pyriproxyfen (1% w/w) and Royal Guard® LLIN manufactured with 5.5 g/kg alpha-cypermethrin (0.55%) and 5.5 g/kg pyriproxyfen (0.55%) were used as the test materials for HPLC method improvement. Extracts from ~ 0.2 g of LLIN were investigated for detection sensitivity using a Vanquish™ Diode Array Detector (VC-D11-A) at shorter wavelengths of 226 and 232 nm compared to the recommended wavelength of 254 nm 15 . The resulting chromatograms are presented in Fig. 3. All three insecticides produced the highest peak heights and corresponding peak areas at 226 nm (Fig. 3). At this wavelength, the greatest sensitivity was recorded for pyriproxyfen with LoD and LoQ of 0.04 µg/ (1 mg/kg net) and 0.1 µg/ (2.5 mg/kg net) respectively, followed by alpha-cypermethrin with LoD and LoQ of 0.06 µg/ (1.5 mg/kg) and 0.18 µg/ (4.5 mg/kg) respectively, and permethrin (cis and trans)) with LoD and LoQ of 2 µg/ (5 mg/kg net) and 0.6 µg/ (15 mg/kg net), respectively. DCP with a retention time well separated from the target insecticides was used as an internal standard to correct for volume errors and to ensure high reproducibility between samples. Four well-separated peaks of pyriproxyfen, DCP, transpermethrin and cis-permethrin were obtained with Olyset® Duo sample (Fig. 3A), and three separat peaks, pyriproxyfen, DCP and alpha-cypermethrin were obtained with Royal Guard® sample (Fig. 3B). An ambient column temperature (23°C) was also used to ensure the method suitability across different laboratory settings. At this temperature, the optimized acetonitrile/water mobile phase ratio 70:30 (v/v), which was slightly higher than the 66.6-33.3 (v/v) recommended method (CIPAC), produced symmetric analyte peaks with no sign of peak abnormalities and clear analyte separation (Fig. 3). Under these conditions the run times for Olyset® Duo and Royal Guard® were 40 min (Fig. 3A) and 30 min (Fig. 3B) respectively compared with 60 min per run in the standard CIPAC method 15 .

Speci city
The improved method was also assessed for method sepeci city to test its ability to measure accurately and speci cally the insecticide of interest in the presence of other components that may be coextracted from the net matrix. Therfore, insecticide peaks determined in both samples were further investigated for the presence of visible interferences (shoulders) by comparison with retention times from insecticide standard injections. Sample retention time of analytes matched the standards with calculated percentage retention times of 100.11% (pyriproxyfen), 100.1% (DCP), 100.23% (trans-permethrin), 100.22% (cispermethrin) for sample extracted from Olyset® Duo (Fig. S1). Similarly, samples extracted from Royal Guard® Net exhibited 100.11% and 100.07% matching retention time for pyriproxyfen and alphacypermethrin, respectively (Fig. S2). In addition, the average peak purities for pyriproxyfen (997), transpermethrin (1000) and cis-permethrin (1000) from sample solutions extracted from Olyset® Duo Net matched the pure analyte peak factor of 1000 (Fig. S1) and for pyriproxyfen (998) and alphacypermethrin (1000) extracted from Royal Guard® Net (Fig. S2).

Linearity
The linearity of the method was examined using a concentration range that encompassed 8 -125% of the target sample concentration for pyriproxyfen, 4% -120% for permethrin and 16 -110% for alphacypermethrin. As presented in Table 2, a linear relationship was obtained between peak area and total concentration of permethrin, alpha-cypermethrin and pyriproxyfen with regression coe cient values close to 1.0 (R 2 > 0.9994). For all tested insecticides, the Y intercepts were effectively zero. The slope agreement was ≤ 5. % relative standard deviation (% RSD) for permethrin, ≤ 2.2% for alpha-cypermethrin and ≤ 0.28% for pyriproxyfen. * Chromatographic conditions used: 70% acetonitrile: 30% water isocratic mobile phase, 1/min ow rate, 40-minute run time and an analysis wavelength of 226nm. The column used for analysis was a Hypersil GOLD C18 column (75 Å, 250 × 4.6 mm, 5-µm particle size; Thermo Scienti c). a Data obtained from linearity validation where b data obtained from LoQ and LoD calculation. A triplicate set of standards were prepared for each insecticide. SD; standard deviation and % RSD; relative standard deviation (SD/Mean*100).

Accuracy and precision
The insecticide recoveries from blank nets forti ed with known quantities of insecticide are presented in Table 3. Permethrin recovery ranged from 101-111%, alpha-cypermethrin recovery ranged from 97.7 -99.4%, while pyriproxyfen recovery ranged from 105-107%. The %RSD was 0.8% for both pyriproxyfen and alpha-cypermethrin and 3.8 for permethrin. Thus, the insecticide recovery for all insecticides examined was close to actual values with high precision. Given the chiral properties of pyrethroids and pyriproxyfen (Fig. 1) and the known vulnerability of pyrethroids to degrade or isomerize upon exposure to light, heat, and solvents 18, 19 , the three insecticides were assessed for their heat stability and resistance to isomerization during extraction. The stability data for permethrin, alpha-cypermethrin and pyriproxyfen before and after heating at 85 o C for 45 minutes are presented in Table 4. The corresponding HPLC chromatograms are shown in Fig. S3, Fig. S4 and Fig. S5 for permethrin, alpha-cypermethrin and pyriproxyfen, respectively. The quantity of the heated standards (permethrin, alpha-cypermethrin and pyriproxyfen) was equal to the unheated standards ( Table 4). None of the examined insecticides demonstrated any signs of degradation/isomerization under the conditions tested (Fig S3, Fig. S4 and Fig. S5). RT; insecticide peak retention time, n; the number of replicates, SD: Standard deviation, %RSD: relative standard deviation (S.D./Mean*100).

Analysis of the total active ingredient(s) content from polyethylene-based LLIN formulations
A range of LLIN formulations (Table 1) were used to evaluate the optimized method as a QCA method for insecticide(s) incorporated into polyethylene-based LLIN formulations and to validate the method reproducibility.

Analysis of LLINs that incorporate a single insecticide
Firstly, to investigate the agreement between the optimized method and CIPAC protocol for the analysis of pyriproxyfen content, a prototype net produced by Sumitomo (Table 1) was analyzed by the optimized method and compared with the standard CIPAC protocol for QCA of pyriproxyfen content in LLIN 15 .
Samples were analyzed in duplicate as recommended by the standard CIPAC protocol 15 and in quadruplet by the new method to account for possible variability in insecticide quantities due to mosaic distribution of a.i. in net material. Graphs comparing data obtained from the two protocols are presented in Fig. 4. The CIPAC method detected 11.25 and 11.7 g/kg for LLIN1 and 2 respectively versus 10.5 and 11.25 g/ kg for the optimized method, which matched the manufactuers target dose 10 ± 2.5 g/Kg. There was no signi cant difference in the average amount of pyriproxyfen extracted from the two nets by either method (P values of 0.68 and 0.87 for LLIN1 and LLIN2 (Fig. 4A) with differences between the two methods close to zero (Fig. 4B).
Next, we assessed the utility of the optimised method to quantify permethrin in Olyset® net, a representative set of standard manufactured LLINs recommended by WHOPES (currently known as PQT-VC) that are incorporated with permethrin at a target dose of 20 g/kg permethrin (2% w/w). To estimate method roubstness and reproducibility for analysis of permethrin content a 24 Olyset® nets were analysed in triplicate. Consistent with WHOPES recommendations 13 , none of the 24 nets scored an average content that differed from that declared by the manufacturer by more than ± 25% (Fig. 5A). Additionally, the method presented a satisfactory level of robustness and reproducibility, as indicated from QCA data shown in Fig. 5B. Out of 24 nets, 23 scored values within +/-2SD of the 18.9 g/kg average while the 21.1 g/kg outlier remains within the WHOPES recommended range 20±5 g/kg. The relative standard deviation (%RSD) of permethrin content was < 10% for all 24 nets analyzed in triplicate (Table  S1), demonstrating the high precession and reproducibility of the HPLC method for permethrin quanti cation.

Analysis of LLINs that incorporate two active ingredients
Twenty four new Olyset® Duo (2% permethrin and 1% pyriproxyfen) were investigated for the simultaneous measurement of pyriproxyfen and permethrin content in LLIN polyethylene polymer following the optimized protocol. None of the 24 nets scored an average dual insecticide content that differed from the amount declared by the manufacture by more than ± 25% (Fig. 6A). The method showed robust reproducibility, as indicated by QCA data (Fig. 6B). All nets scored values within +/-2SD of the average of 19.1 ± 1.3 g/kg for permethrin and 10.4 ± 0.5 g/kg for pyriproxyfen (Fig. 6B). An indicative of the high precision of the HPLC method, the relative standard deviation (%RSD) of permethrin and pyriproxyfen content obtained from all samples analyzed in triplicate was less than 10% (Table S2).
To establish the broader applicability of the new method for next-generation LLINs that are commercially available for malaria control, thirty Royal Guard ® Net containing a mixture of alpha-cypermethrin and pyriproxyfen were assessed for insecticide content. None of the 30-nets scored an insecticide content that differed from the declared manufacturer's 5.5 g/kg concentration by more than ± 25% (Fig. 7). However, a slight increase in the alpha-cypermethrin content has been noted, giving a value of 6.03 ± 0.33 g/kg (Fig. 7B).
The manufactured loading of active ingredient contents was further investigated by taking a random net from the 30 nets and subjecting it to ve cycles of insecticide extraction in triplicates. The majority of the active ingredients were extracted in the rst run (Fig. 6S). Pyriproxyfen quantity recovered in the rst round of the extraction was 5.4 ± 0.46 g/kg and alpha-cypermethrin quantity was 5.6±0.14 g/kg, which is approximately equivalent to the manufacturer's reference value for both insecticides (5.5 ± 1.375 g/kg) (Fig. 6S). Compared to the rst run, a negligible amount of the two active ingredients were recovered in the subsequent four runs, amounting to a residual amount of 0.02 and 0.6 g/kg of pyriproxyfen and alpha-cypermethrin likely carried over from the rst run (Fig. 6S).
To test the method reproducibility, ten samples from one Royal Guard ® net were analyzed using the optmised protocol. Again, the method scored values similar to the manufacturer's average, 5.75 ± 0.16 and 5.43 ± 0.34 g/kg of pyriproxyfen and alpha-cypermethrin respectively (Fig. 7S). Analysis of ten samples with the optmised method also represented high reproducibility with %RSD < 6.2 for both active ingredients.

Discussion
We have developed a simpli ed approach for sample preparation, extraction and insecticide quanti cation from LLINs made from polyethylene polymers that incorporate pyrethroid and pyriproxyfen insecticides. The standard CIPAC protocol for the QCA of pyriproxyfen net recommends heating large amounts of net material (~ 2g) with 50 of the solvent mixture at 85-90°C in duplicate, which results in the production of a signi cant amount of solvent waste that if scaled for multiple nets could be problematic for public health and the environment 17,20,21 . Solvent selection guideline has identi ed heptane as a problematic but not hazardous solvent 17,21 . By reducing the sample size to ~0.2 g we were able to reduce the solvent used for extraction by 10-fold, providing greener chemistry and sustainable solvent use in chemical processing, and eliminating the need for rotary evaporation that prevents the facile evaporation of multiple samples for high throughput analysis of multiple LLINs. Chromatographic conditions were also optimized for the separation and quantitation of pyriproxyfen, permethrin and alpha-cypermethrin. The U.V. detection wavelength of 226 nm and mobile phase composition of 70% acetonitrile in water has helped to achieve higher sensitivity for insecticide detection and quanti cation with the small sample size (0.2 g) at shorter 30 -40 min run time relative to CIPAC (60 min) 15 .
The extraction and recovery of additives incorporated into a plastic polymer can be also di cult and usually requires the complete dissociation and solvation of the polymer material using hazardous solvents such as xylene at high temperature (>140°C). With our protocol, heating LLINs with heptane at 85°C for 45 min was su cient to recover insecticides (permethrin, alpha-cypermethrin and pyriproxyfen) from the polyethylene bers by swelling of the polymer without dissolving the bre. Similarly, iso-octane has been tested previously as a universal solvent for pyrethroid extraction from polyester and polyethylene nets without dissolving bre 16 . However, the extraction was reliant on large sample size and lacked an internal standard 16 , thus prone to variability in insecticide quanti cation due to solvent volatility. In contrast, our method doesn't preclude the internal standard (DCP) recommended in the original CIPAC protocol 15 , resulting in a more robust and reproducible method for the quantitative analysis of the active ingredients from LLINs ( Fig. 5-7).
The new method facilitates the analysis of insecticides by enabling multiple net samples to be processed in parallel using standard low volume tubes and multiwall dry blocks for solvent evaporation (Fig. 2). Figure 1 Chemical structure of permethrin, alpha-cypermethrin and pyriproxyfen insecticides (*: chiral centres).

Figure 2
Comparison of standard CIPAC method with a miniaturised protocol for determining insecticide content incorporated in long-lasting bed nets (LLINs). The sample size has been reduced from 400 cm 2 (2 g) tõ 40 cm 2 (0.2 g) to enable a small volume of extraction solution (5 vs 50 used in the standard CIPAC methods) for permethrin 14 and pyriproxyfen 15 respectively.  ). An unpaired t-test was used to calculate the signi cant difference between the two methods at the p-value of 0.67. ns; no signi cance.