In-house calibration of UHPLC for determination of aflatoxins in groundnuts and red chili

doi:10.21203/rs.3.rs-4847512/v1

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In-house calibration of UHPLC for determination of aflatoxins in groundnuts and red chili

https://doi.org/10.21203/rs.3.rs-4847512/v1

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Routine internal calibration is necessary to evaluate the performance of ultrahigh-performance liquid chromatography (UHPLC); therefore, false negative and false positive results are avoided. Herein, we report a detailed internal calibration protocol and validation procedure for the determination of aflatoxins in groundnuts and red chili plants via UHPLC to comply with the requirements of ISO/IEC: 17025. The validated parameters included the flow rate accuracy, injector accuracy, temperature accuracy, system precision, photodiode detector, detector linearity, injector linearity, gradient proportion test, fluorescence detector linearity and wavelength accuracy results. The obtained percent deviation, relative standard deviation and r²values for injector accuracy, system precision and calibration curves were -0.77, 0.78 and ≥ 0.999, respectively. During the development of the aflatoxin analysis scope, internally calibrated UHPLC was used for separating the aflatoxin mixture at a concentration of 5 µg/kg in the ground nut and red chili matrices. Witness test results revealed that the quantification of individual aflatoxins at 0.5 µg/kg in the red chili matrix was achievable. A stability assessment of the aflatoxin standards solubilized in the solvent was achieved by generating control plots, and uncertainties were also measured.

Calibration

Linearity

Precision

Aflatoxin

Quality control

UHPLC

Aflatoxin testing under the most important fields of agricultural sciences and engineering is recommended by the National Accreditation Board following the ISO/IEC:17025 standard. To achieve this goal, periodic internal and external calibrations are necessary for highly sensitive equipment, such as high-performance liquid chromatography (HPLC), liquid chromatography‒mass spectrometry (LC‒MS), gas chromatography‒mass spectrometry (GC‒MS) and inductively coupled plasma‒mass spectrometry (ICP‒MS), as suggested in the international procedure for the validation of bioanalytical methods [1-3]. In any measurement process, analytical variation occurs from the presence of random errors during sample preparation originating from the presence of interfering compounds in the matrix [4, 5]. Analytical laboratories need the most reliable and robust equipment to ensure product consistency and compliance with the regulatory guidelines and quality standards of Good Manufacturing Practice (GMP), Good Laboratory Practice (GLP), ISO/IEC 17025:2017 and Quality Policy. The integrity of the method is always maintained during the transfer of methods across the organization or contractual firms. In general, the overall laboratory performance depends on the accuracy, timeliness and reliability of the test results generated via analytical equipment. Thus, quality assurance in the testing laboratory includes proficiency testing, interlaboratory comparisons, retesting, replicate testing and, ultimately, equipment calibration.The current introduction of UHPLC equipment in laboratories has paved the way for novel method development and analysis of newer pharmaceuticals and various biochemical compounds [6, 7]. The equipment was calibrated against an internal standard (IS) by comparing the instrument responses of the reference standards to the responses of the target compounds. Equipment reliability is the ultimate need of the testing laboratory

Numerous approaches include determining the quantity of internal standard used in the preparation of standards and samples; however, no definitive results have been obtained [8, 9]. The peak height or area of the internal standard was comparable to that of the analyte of interest, but supporting data are needed for a vivid interpretation. The experimental strategies that can be used to verify the optimum amount of internal standard were described by Araujo et al. [10]. There are reports that internal standards should be used at the highest possible concentration [11]. The response factor calculation revealed that the detector provides a linear response for both the analyte and the internal standard over the entire range of the experiment. It is necessary to determine the behavior of the response factor because the concentrations of the analyte and internal standard are different. Understanding the behavior of the response factor limits the range of combinations of analytes and internal standards at different concentrations [12, 13].

The Internal calibration is utilized to increase the precision and accuracy of the results when errors due to volume are difficult to estimate and manage [14, 15]. The types of errors are reduced by the use of an internal standard. Errors occur during solvent evaporation, injection, complex sample preparation, extraction and dilution. An internal standard must be selected accurately, and a known amount must be spiked cautiously into both the sample and standard solutions to prevent miscalculations. The resulting internal standard peak must be resolved well from other components in the sample and appropriately incorporated. If the stipulated conditions are not satisfied, the usage of an internal standard may, in reality, add to the inconsistency in the outcome [16, 17]. It is possible that when the detector noise or the integration errors are the overriding reasons for the error, the employment of an internal standard will probably make the experimental results more futile. In our effort, a methodical approach was used to conduct internal calibration and improve precision following an internal standard method. The data were recorded at numerous injection volumes with two different standards. We developed an ultrahigh-performance liquid chromatography method to quantify a panel of four aflatoxin isomers in red chili powder via an internal calibration approach. Moreover, the applicability of the methods was assessed by analyzing commercial, fresh farm and retail shop products.

Chemicals

Caffeine (purity 99.96%) and anthracene (purity 99.0%) reference materials were obtained from Sigma Aldrich. HPLC grade methanol was purchased from Merck. HPLC-grade water was obtained from Merck Millipore water purifier (18.2 MΩ.cm). The calibrated volumetric flasks of Grade A were purchased from Borosil. Nylon membrane filter papers (47 mm, 0.22 µm) for filtration of the mobile phase solutions were purchased from Avantor Lab Supplies, India.

Instrumentation

An ultrahigh-performance liquid chromatography (NexeraX2, UHPLC) system (Shimadzu, Japan) was assembled with an SIL-30AC autosampler, a DGU-20A 3R degassing unit, an LC-30AD binary pump, a CTO-20 AC column oven with a maximum temperature of up to 90°C, an SPD-M20A detector and an RF-20A detector. The calibration of the equipment leaving the RF-20A detector was performed with a caffeine standard via a Purospher STAR RP 18e (250 mm × 4.6 mm, 5 µm) column at 27°C using mobile phases A: water (30%) and B: methanol (70%) at 0.7 mL min^-1 and a 20 µL injection volume. A wavelength of 272 nm was selected for the detection of the caffeine peak on the PDA detector. The fluorescence detector was calibrated against an anthracene standard via a Phenomenex Luna C18 (250 mm × 4.6 mm, 5 µm) column at 30°C using mobile phase water (20%) and B: methanol (80%) at 1.0 mL min^-1 in a 2 µL injection volume. The excitation and emission wavelengths were selected to be 360 nm and 450 nm, respectively, for the detection of the anthracene peak on the RF detector. Lab Solutions software version 5.60 SP2 was applied for the automatic processing of chromatographic peaks for each of the parameters calibrated.

Equipment calibration

Flow rate accuracy

The flow rate accuracy of both pump modules (A/B, NexaraX2, and LC-30AD) was characterized to assess the proper pumping of the mobile phase solution (HPLC grade water) at flow rates of -0.2, 0.4, 0.6, 0.8, 1.0 and 2.0 mL min^-1. The procedure involved recording the empty mass of a 10 mL calibrated volumetric flask (m₁), after which the flask was inserted with column inlet tubing to collect the mobile phase solution for two minutes at each flow rate separately via a stopwatch. The mass of the water in the volumetric flask (m₂) was recorded and recorded. The deviation was calculated from the density of water (ρ) at 25°C via the following formula. Error flow was calculated by subtracting set flow values from actual flow values.

Vial test

The efficiency of the autoinjector was tested for its movement in an autosampler by taking five empty HPLC vials with new septa and placing them at different vial positions in the autosampler vial from racks no. 1 and 0. The respective vial positions were given commands through the software, and the batch was started.

Injector accuracy

The empty weight of the clean HPLC vial with a cap was determined, and the mass (m₁) of the vial filled with water was recorded. A batch table was created, and the system was run six times (n = 6) with HPLC-grade water as the mobile phase for a 50 µL injection volume. The weight after six injections was recorded (m₂), and the mass of the injected volume was calculated. Injector accuracy was verified by calculating the deviation of the injected volume. The deviation was calculated by dividing the mass after injection by the mass of the injected volume.

Accuracy of the column oven temperature

The actual working temperature range of the column oven was 30°C–60°C, and the temperature was calibrated via a digital thermometer. The column oven was allowed to stand for 10 min at the set temperature, after which the analytes were measured via a digital thermometer at 30°C, 45°C and 60°C. The deviation in temperature was calculated and tabulated.

System precision

The precision of the UHPLC system was determined via the mobile phase (methanol–water [7:3, v/v]) by connecting the mixture to a Purospher STAR RP C18 (250 mm × 4.6 mm, 5 µm) column. The caffeine standard (10 mg) was accurately weighed and dissolved in 10 mL of methanol to a stock concentration of 1000 µg mL^-1. From the stock, a caffeine solution with a concentration of 60 µg mL^-1 was prepared by transferring 600 µL of the solution into 9.4 mL of methanol, after which the mixture was repeatedly injected into the system six times at a 20 µL injection volume. Chromatograms were integrated, and the RSD was calculated for the retention time and the peak area within a limit [18].

Wavelength accuracy

The caffeine standard signal obtained by a PDA detector at different wavelengths was correlated with the area of the standard at each wavelength. The equipment was conditioned with a mobile phase consisting of methanol: water (7:3 v/v) after being connected to a Purospher STAR RP C18 column (250 mm × 4.6 mm, 5 µm). Briefly, 20 µL of 60 µg mL^-1 caffeine solution was prepared and injected (n = 3). The chromatograms obtained were integrated at different wavelengths ranging from 202–208 nm and 269–275 nm, with an increase in the 1 nm interval. The average area of the peak at each wavelength was recorded [19].

Caffeine PDA detector linearity

A six-point working standard linearity was drawn to correlate the linear response of the PDA detector with that of the linearly increased standard concentrations. The equipment was conditioned with the mobile phase system methanol: water (7:3, v/v) in the presence of a Purospher STAR RP C₁₈ column (250 mm × 4.6 mm, 5 µm). Linear concentrations of caffeine in methanol (6, 12, 24, 30, 48 and 60 µg mL^-1) were prepared from the stock solutions. Calibration standards of a 10 µL injection volume were used in three replicates (n = 3). The obtained chromatograms were linearly related, and the r² values were recorded [20].

Caffeine injector association

The linearity of the injector was determined via the mobile phase (methanol-H₂O, 7:3, v/v) by connecting it to a Purospher STAR RP C₁₈ (250 mm × 4.6 mm, 5 µm) column. Caffeine solution (60 µg mL^-1) was prepared from the stock in methanol. A batch table with linear injection volumes of 1, 5, 10, 15, 20 and 25 µL (n = 3) was created and started.

Gradient proportion valve test (GPV)

The gradient mode of both pumps was tested in the presence of caffeine solution at different proportions. The equipment was conditioned with a mobile phase consisting of water (A) and caffeine solution (10 µg mL^-1) (B) with a union joint at a flow rate of 1 mL min^-1 and 272 nm in gradient mode as described in the Shimadzu Operational Qualification (OQ) document [21]. The mobile phase solution was passed through the union joint starting from 0–100% B for 30 min. The heights of the bands were integrated, and the deviations were calculated at the 10, 50 and 90% levels via the gradient proportion method [22].

Anthracene fluorescence detector linearity

The additional fluorescence detector used was calibrated to evaluate the correlation coefficient for the anthracene standard with the mobile phase combination of methanol: water (8:2, v/v) via a Phenomenex Luna C₁₈ (250 mm × 4.6 mm, 5 µm) column. Working standard concentrations of 5, 10, 15, 20, 25 and 30 µg mL⁻1 anthracene in methanol were prepared by pipeting 5, 10, 15, 20, 25 and 30 µL solutions from a 1000 µg mL^− 1 stock. A batch table was created, and each solution (2 µL) was injected into the equipment (n = 3). After the chromatograms were integrated, the linearity was determined, and the r² value was recorded [23–25].

Calibration is a set of operations under specified conditions that establish the relationship between the values shown by the instrument and the corresponding known values of the measurand. Internal calibration can be performed when suitable analytes with physiochemical properties analogous to those of authentic analytes are available [26]. Errors in the testing facility can occur in three different test stages, i.e., preanalytical, analytical and postanalytical. Analytical stage errors arise during the process of testing by using noncalibrated equipment or by the use of incorrect proportions of reagents and nonadherence to the standard operating procedure. The parameters used in this calibration work were flow rate accuracy, vial test, injector accuracy, oven temperature accuracy, system precision (RSD), wavelength accuracy (nm), detector linearity (r²), injector linearity (r²), gradient proportion valve test (% B Conc.) and fluorescence detector linearity (r²). The flow rate error is calculated for pump modules A and B at a ratio of 1:1.

Further values were tabulated, and the error was within the limits. The error in the flow rate was ± 0.0, which indicated that the flow rate was accurate for both pumps A and B (Table 1). Our novel advances require supplementary initial experiments to evaluate the linearity and stability of the analyte and to evaluate the limit of quantification [27]. As part of the maintenance, the vial test parameter was needed to indicate whether the injector needle was moving in the right direction. The performance of the samples from the vial test was recorded, and the septa were pierced. This test confirmed that the vials were pierced and that flow was established.

The introduction of a sample inside the UHPLC system is crucial. The protection of the UHPLC columns from severe pressure fluctuations is managed when the injection process is relatively pulse-free. The swept volume of the device is assumed to be negligible to reduce probable band spreading [28]. Typically, a rapid injection cycle time is considered essential to fully exploit the speed provided by UHPLC, which in turn requires a high sample capacity. Low-volume injections with minimal carryover are required to increase the sensitivity. There is also a direct injection approach for introducing biological samples [29]. The accuracy of the injector of the NexeraX2 SIL-30AC system was calculated by injecting exactly 50 µL of HPLC-grade water as a blank six times, and the deviation was found to be -0.77 µL, which was within the criterion of ± 1.0 µL. This was found to be satisfactory. For many applications, the accuracy of the flow rates of mobile phases in HPLC systems is not critical; hence, the need for traceable calibration to national or international standards is less important. In such cases, the accuracy of the operating parameter is secondary, provided that it remains consistently reproducible during the analysis of both the sample and the standard. However, in other cases, accuracy is needed when a developed analytical procedure is transferred to another laboratory for routine use [30].

Table 1

Flow rate accuracy of the pump at different set flow rates.
Sl. No.	Set flow (mL min^-1) (A/B 1:1)	Mass of the empty flask (m₁, g)	Mass of the filled flask (m₂, g)	Time (min)	Water density (g/cm³)	Actual flow rate (mL min^-1)	Error flow rate (mL min^-1)	Criteria (mL min^-1)
1	0.2	25.1436	25.5452	2	0.997	0.2014	0.0014	± 0.01
2	0.5	24.7054	25.7010			0.4993	-0.0007	± 0.01
3	1.0	24.8598	26.8534			0.9998	-0.0002	± 0.02
4	2.0	24.2754	28.2376			1.9871	-0.0129	± 0.04

The UHPLC CTO-20AC oven accuracy was calibrated and verified with a calibrated digital thermometer. The actual temperature settings given to the system through the software were verified after 10 min of stabilization via a calibrated digital thermometer. The actual and observed temperatures were recorded, and the deviation in the temperature was within ± 1.0°C; these observations fulfilled the criteria.

First, the weights of the analytes were varied against a constant internal standard concentration, and later, the concentration of the internal standard was varied against a constant weight of the analyte. The standard deviation was chosen to monitor the precision, and the system precision was evaluated by injecting 20 µL of caffeine standard solution (60 µg mL^-1, n = 6) with methanol-water (70/30, v/v) as the mobile phase. The RSDs for the peak area and retention time were determined to be 0.78% and 0.08 min, respectively (Table 2).

Table 2

System precision using the caffeine standard of UHPLC
Sl. No.	Replicate (n)	RT (min)	Peak area	Criteria (RSD)
1	R1	4.843	2386466	≤ 1.0
2	R2	4.838	2368715
3	R3	4.841	2413959
4	R4	4.833	2378476
5	R5	4.838	2413902
6	R6	4.834	2398159
	Mean	4.838	2393280
	SD	0.004	18685
	RSD	0.08	0.78%

The wavelength accuracy of the system for caffeine standard solution (60 µg mL^− 1) was tested in triplicate at wavelengths ranging from 202–208 nm and 269–275 nm. The maximum responses in terms of peak areas of the caffeine standard were recorded. The wavelength accuracy of the detector suggested that caffeine had a maximum response at wavelengths of 204 and 271 nm.

Table 3

Wavelength accuracy of the SPD-M20A detector at different wavelengths (n = 3).
Sl. No.	Wavelength (nm)	R1	Response(Area) R2	R3	Avg. Response (area) (n = 3)	Criteria (nm)
1	202	5971402	5958326	5960331	5963353	205 ± 2
2	203	6003976	6080956	6126503	6070478
3	204	6254035	6037720	6056821	6116192
4	205	6007447	6143645	6182075	6111056
5	206	6191494	5967989	6162123	6107202
6	207	5930416	6198636	5935415	4516169
7	208	5958961	5740640	5864715	5854772
8	269	2327108	2320457	2247623	2298396	272 ± 2
9	270	2370025	2383426	2381928	2378460
10	271	2361317	2386225	2360031	2369191
11	272	2394946	2298979	2379875	2357933
12	273	2384404	2388111	2334571	2369029
13	274	2438180	2419905	2526355	2461480
14	275	2408631	2391201	2404331	2401388

The detector linearity was evaluated by injecting the caffeine standard solution (20 µL) in triplicate. The linearity of the detector was determined by taking the peak area versus the concentration, and an r² value of 0.9997 was obtained (Fig. 1). This was found to be satisfactory.

The linearity of the injector (SIL 30AC) was evaluated at six different injection volumes of caffeine standard (60 µg mL^− 1), after which the standard curve was plotted (Fig. 1). The correlation coefficient of determination was reported as r² = 0.9993. In our experiment, detector linearity was assessed with respect to the anthracene standard solution because it is a colorless compound but exhibits blue fluorescence under ultraviolet radiation. The absorption of the caffeine standard ranged from 202–208 nm to 269–275 nm, although the maximum response of the caffeine standard was recorded at wavelengths of 205 nm and 271 nm. This standard efficiently establishes injector linearity, and the results of this study coherently revealed that good precision occurs when the volume of the injection mixture is 1–25 µL of caffeine solution at a concentration of 60 µg mL^− 1. However, accurate responses were observed with increasing injection volume. In general, injector linearity and injection-to-injection variation have distinct effects on precision when the injection volume is reduced to the smallest quantity. Therefore, an injection volume of 1–25 µL was sufficient for reducing errors, and there was no need for the existing method to be upgraded. This approach holds good for any new external or internal standards that are used in this method.

The core of the performance test is a check of the system’s ability to create accurate linear gradients. HPLC-grade water was placed in reservoir A, and water containing 0.1% acetone was placed in reservoir B. The column was removed and replaced with approximately 1 m of 0.005 in. i.d. tubing. The detector wavelength was set to 265 nm, and the flow rate was set to 1–2 mL/min to create sufficient pressure for reliable check valve operation.

A gradient concentration test was performed to evaluate the proper function of the pump at the adopted gradient concentration. HPLC-grade water was placed in solvent reservoir A, and water containing caffeine was placed in reservoir B. A series of 5 min steps were run in 10% increments (0% B, 10% B, 20% B… 100% B). The calculated percentages and their deviations were as follows: tabulated (Fig. 3 and Table 4). The % concentration deviation at each level ranged from 0.28 to 0.4% and the passing results were generally ≤ 1%. Therefore, the results passed the criteria of ± 1.00%, as the deviation at each level should be ± 1.0%. The difference between the maximum and minimum plateau heights must be less than 5% of the average height. A graphical representation of the gradient step test results is shown in Fig. 3. The steps look good and appear flat with slight rounding at the edge of each step. The step size was calculated for each step and compared to the theoretical (programmed) value. The largest error was 0.4%, which was less than the 1% allowable maximum; thus, the step test was passed under the initial conditions (Table 4). This procedure tests all combinations of the proportioning valves for accurate delivery. Although a 5% limit was given, the passing results were generally ≤ 1%, confirming good general pump performance, no blockage of inlet line frits and no proportioning-valve failure.

Table 4

The % B deviation at different concentrations.
Conc. (%B)	Height of band	Conc. (%)	Criteria (%)	Error (%)
0	0.00	--	± 1.0
10	36135	9.96		0.40
50	180843	49.86		0.28
90	325143	89.65		0.39
100	362687	99.55		0.39

Analyte detection is typically based on the absorbance produced by the analyte and is directly proportional to the concentration and sensitivity of the detector. Typically, UHPLC has a reduced flow cell volume to maintain both the concentration and the signal. In accordance with Beer–Lambart’s law, small-volume conventional flow cells are adopted, resulting in a reduction in path length and thereby strengthening of the signal [31]. The linearity of the fluorescence detector at various concentrations (5–30 µg/mL) was calibrated with 2 µL of anthracene solution (n = 3) as a standard, and the standard curve was plotted with r² = 0.9990 (Fig. 4).

The calibrated UHPLC equipment was used for determining the scope of aflatoxin analysis from matrices such as red chili, groundnut and nut products. To further determine the accreditation status for the developed scope by the National Accreditation Board for Testing and Calibration Laboratories (NABL), an internal assessment was conducted by the board, which included the verification of the in-house method and the validation of the method specific to the matrices applied for accreditation. The results of the witness tests for aflatoxin isomers G1, G2, B1 and B2 in the chili matrix clearly revealed separation of all four molecules at a spike level of 0.5 µg/kg (Fig. 5), and the results of the witness test revealed detection of up to 0.5 µg/kg individual aflatoxin and recovery within the range of 70–119%. Nevertheless, attention must be given to the injection volume and the manual errors that are caused during the addition of the internal standard to the analyte.

As the Aflatoxin mixture stock (1 mg mL^− 1) was stored at -20°C for a longer period during the experimental procedure, the stability of the stock was assessed for ten months at a concentration of 100 µg mL^− 1 by monitoring the reference material control charts developed by using the internally calibrated UHPLC. The control plots were drawn once every two months to ensure the stability of the standard [32]. The charts (Fig. 6) were constructed by taking the average peak area of replicates (n = 3) with their standard deviation (+ 3 and − 3 times the standard deviation (SD) reported and found to be acceptable for all the aflatoxin molecules over a period of ten months [33]. In Fig. 6, green indicates the upper control limit, and blue indicates that the standard response to aflatoxin was satisfactory. The results showed that the standard mixture of aflatoxin did not cause any degradation.

The uncertainty (MU) (± ng g^− 1) was calculated for the aflatoxins AFB₁, AFB₂, AFG₁ and AFG₂ in the red chili powder matrix at three recovery batch levels by computing the associated uncertainties due to factors such as reference material impurities, analytical balance, calibrated volumetric flasks, micropipettes and averaged area responses [34].The MU of the final result obtained by combining the uncertainties of the independent inputs with the same rule used is the square root of the sum of the squares, but the choice of using the standard deviation (SD) or coefficient of variation (CV) depends on whether the inputs interact by addition or subtraction or by multiplication or division. The reported uncertainties for the four aflatoxin residues at each level (Table 5) were 2.5 ± 0.4.5 ng g^− 1, 5 ± (0.89–1.15) ng g^− 1, and 10 ± (2.10–2.56) ng g^− 1.

Table 5

Measurement of uncertainty in the determination of AFB₁, AFB₂, AFG₁ and AFG₂ in red chili powder, three different quantification limits were used.
Aflatoxin	(2.5 ng g^− 1) 1 LOQ	(5.0 ng g^− 1) 2.5 LOQ	(10.0 ng g^− 1) 5 LOQ
AFB1	2.26 ± 0.54	5.13 ± 0.94	10.48 ± 2.29
AFB2	2.24 ± 0.52	5.03 ± 0.91	10.36 ± 2.24
AFG1	2.12 ± 0.42	4.90 ± 0.89	9.86 ± 2.10
AFG2	1.95 ± 0.35	4.05 ± 1.15	10.82 ± 2.56

This paper describes the guidance for the internal calibration of UHPLC and provides documented evidence of whether the instrument is fit for aflatoxin analysis. Reproducible quantification of aflatoxin was performed on groundnut and red chili matrices during method validation and witness tests. The following parameters passed the competency test: flow rate accuracy (1), vial test (2), injector accuracy (3), oven temperature accuracy (4), system precision (5), wavelength accuracy (6), detector linearity (7), injector linearity (8), gradient proportion valve test (9), and fluorescence detector linearity (10).These parameters are critical because their performance determines the calibration status of the equipment. In conclusion, the laboratory must be calibrated internally at least once a year to account for the errors caused by volume fluctuations, injection-to-injection variations, volume changes during sample preparation and variations in peak responses.

Conflict of interest

The author and coauthors declare that they have no conflicts of interest.

Author Contribution

SNR has conceptualized the idea, designed the experiments and written the manuscript.CRV, SP and MK have worked at the bench level.NM and AP have edited the manuscript .

Acknowledgment

The authors would like to acknowledge the University of Agricultural Sciences, Raichur and Rashtriya Krishi Vikas Yojana (Ab. Ac. No. 6789) Ministry of Agriculture, Govt. of India for financial support.

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In-house calibration of UHPLC for determination of aflatoxins in groundnuts and red chili

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Chemicals

Instrumentation

Equipment calibration

Flow rate accuracy

Vial test

Injector accuracy

Accuracy of the column oven temperature

System precision

Wavelength accuracy

Caffeine PDA detector linearity

Caffeine injector association

Gradient proportion valve test (GPV)

Anthracene fluorescence detector linearity

Results and Discussion

Conclusions

Declarations

Conflict of interest

Author Contribution

Acknowledgment

References

Additional Declarations

Supplementary Files

Status:

Version 1