CN110596229A - Detection method for element impurities in hemp extract and hemp oil product - Google Patents

Abstract

The invention relates to a method for detecting element impurities, in particular to a method for detecting element impurities in a hemp extract and a hemp oil product. The method specifically comprises the following steps: selecting an analysis method: using inductively coupled plasma mass spectrometry; reference standard: selecting standard arsenic, cadmium, lead and mercury products with concentration of less than 1000 mg/L; reagent solvent: selecting concentrated nitric acid, ultrapure water and 100% ethanol; wherein the mass concentration of the concentrated nitric acid is 68-97.5%; selecting a sample: selecting cannabis extract and cannabis oil; preparing standards, internal standards, blanks and samples; and respectively testing according to different verification parameters. The test method of the present invention provides clear evidence for reliable and consistent results within the detection and analysis range of elemental impurities As (75), Cd (114), Hg (202) and Pb (208) (10-300% of the working standard concentration) in cannabis extracts and cannabis oil products.

Description

Detection method for element impurities in hemp extract and hemp oil product

Technical Field

The invention relates to a method for detecting element impurities, in particular to a method for detecting element impurities in a hemp extract and a hemp oil product.

Background

The metal elements have high toxicity and long latent period, can be enriched along the food chain and have obvious harm to human body. Metal elements entering various environments such as atmosphere, water body and soil can be absorbed by animals through various ways such as respiratory tract, digestive tract and skin, and when a certain amount of metal elements are accumulated, the metal elements can damage a nervous system, a hematopoietic system and a digestion system, so that the immunity of the organism is reduced.

In recent years, the problems of dissolution detection of metal impurities and the limit thereof are more and more emphasized at home and abroad, and standards and guiding principles aiming at the limit of metal elements, such as United states Pharmacopeia <232 limit of impurity of medicine elements >, ICH Q3D < guiding principle of impurity of elements >, and guiding documents specified by the limit of residual quantity of metal catalysts or metal reagents of EMEA/CHMP, are continuously provided internationally. However, the prior art has no safe and reliable detection method for the element impurities in the hemp extract and the hemp oil product.

CN107205960A (2017-09-20) discloses a cannabis extract and its preparation and use methods, however, does not relate to the detection method of the elemental impurities therein;

CN110004179A (2019-07-12) discloses a general detection method for cannabinoids active substances and a detection kit thereof, however, the detection method for element impurities in the cannabinoids active substances is not involved.

CN104597110A (2015-05-06) discloses an ICP-MS determination method of trace element impurities in high-purity chromium, but is not suitable for detecting element impurities in hemp extracts and hemp oil products.

Disclosure of Invention

The invention aims to provide a method for detecting element impurities in a hemp extract and a hemp oil product.

The technical purpose of the invention is realized by the following technical scheme:

a method for detecting element impurities in a hemp extract and a hemp oil product comprises the following specific steps:

(1) selecting an analysis method: mass spectrometric detection using inductively coupled plasma

(2) Determination of the reference standard: selecting standard arsenic, cadmium, lead and mercury products with concentration of less than 1000 mg/L;

(3) selecting a reagent solvent: selecting concentrated nitric acid, ultrapure water and 100% ethanol; wherein the mass concentration of the concentrated nitric acid is 68-97.5%;

(4) selecting a sample: selecting cannabis extract and cannabis oil;

(5) preparing standards, internal standards, blanks and samples;

(6) the test was performed according to the following verification parameters:

the system applicability is as follows;

b, quantitative limit value and detection limit value;

c, testing accuracy: comprising i) testing the accuracy of cannabis oil;

ii) accuracy testing of cannabis extracts;

d, testing the range;

e, testing the accuracy: comprising i) a reproducibility test for cannabis oil;

ii) reproducibility testing of the cannabis extracts;

iii) intermediate precision testing;

and F, testing the specificity.

The purpose of this method is to provide evidence that this test method can produce reliable and consistent results over the analytical range of As (75), Cd (114), Hg (202) and Pb (208) (10-300% of the working standard concentration).

Inductively coupled plasma mass spectrometry (ICP-MS) exploits the ability of ICP to generate charged ions from elemental species in a sample. These ions are then introduced into a mass spectrometer, which separates them according to their mass-to-charge ratios. The content of elemental impurities should be limited and different limits may be provided depending on the daily exposure. The ICP-MS (inductively coupled plasma mass spectrometry) method has the advantages of low detection limit, less interference, wide dynamic linear range and high analysis precision, and can meet the requirement of multi-element simultaneous determination.

Preferably, the quantification limit is used to test a quantifiable minimum level; quantification limit the detection limit is determined by accuracy for detecting the concentration of the analyte.

Preferably, the quantification limit is equal to 8-12 times the standard deviation of the blank reagent.

More preferably, the quantitation limit is equal to 10 times the standard deviation of the blank reagent.

More preferably, the calculated quantitative limit is below the lowest concentration level obtained in the accuracy study.

Preferably, the detection limit is equal to 2-4 times the standard deviation of the blank reagent.

More preferably, the detection limit is equal to 3 times the standard deviation of the blank reagent.

Preferably, the detection limit is equal to the non-invasive micro-measurement technique multiplied by a specification quantitation limit concentration of 0.05-0.15 times.

More preferably, the detection limit is equal to the non-invasive micrometric technique multiplied by 0.1 times the specification quantitation limit concentration.

Preferably, the accuracy test is used to test how close a found amount is to a known added amount; triplicates were prepared at 50%, 100%, 150% of the limit; the average recovery per grade was 70-150%.

Preferably, the range test is used to test the linear response range.

Preferably, the accuracy test is performed in particular 6 or more repetitions at the 100% limit.

Preferably, the target element does not float more than 20%.

More preferably, the standard calibration float range in system applicability is-5.2% -5.9%.

Preferably, the accuracy studies are performed at 10%, 50%, 100%, 150% and 300% of the limiting concentrations of As (75), Cd (114), Hg (202) and Pb (208), respectively.

More preferably, the accuracy of the elemental impurity As (75) is in the range of 1.2ppb to 36 ppb; accuracy of Cd (114) and Pb (208) is 0.4ppb to 12 ppb; the accuracy of Hg (202) is 0.8ppb to 24 ppb.

More preferably, the acceptance criterion for intermediate cannabis extract precision is 20% -25% NMT per target element RSD.

Preferably, the preparation method of the standard sample of the hemp extract comprises the following steps:

1) accurately weighing 0.1-0.9g of cannabis sativa extract into a 50mL centrifuge tube;

2) adding 0.5-3mL of ethanol to each tube; each tube was then capped and vortexed until completely dissolved;

3) transferring nitric acid, hydrochloric acid, intermediate standard-2 solution, 150-;

4) then, tightly covering each tube, uniformly shaking by hand, and transferring the sample into a digestion tube;

5) digesting all samples in a multi-band GO digestion system; digestion time: 5-15 minutes; target temperature: 120 ℃ to 210 ℃; retention time: 15-25 minutes;

6) after digestion, transferring the sample into a centrifuge tube; rinsing the digestion tube with ultrapure water, and combining rinsing liquids into the same centrifuge tube; each tube was diluted to 50mL mark with ultrapure water, capped and mixed well by hand.

More preferably, the method for preparing a standardized sample of cannabis extract comprises the steps of:

1) accurately weighing 0.1-0.9g of cannabis sativa extract into a 50mL centrifuge tube;

2) add 1mL of ethanol to each tube; each tube was then capped and vortexed until completely dissolved;

3) transferring nitric acid, hydrochloric acid, intermediate standard-2 solution, 200 μ Ι _ internal working standard solution and 50 μ Ι _ gold standard into the same centrifuge tube, the cover tube is loose and allowed to cool in a fume hood;

4) then, tightly covering each tube, uniformly shaking by hand, and transferring the sample into a digestion tube;

5) digesting all samples in a multi-band GO digestion system; digestion time: 10 minutes; target temperature: 120 ℃ to 210 ℃; retention time: 15-25 minutes;

6) after digestion, transferring the sample into a 50mL centrifuge tube; rinsing the digestion tubes with Milli-Q water and combining the rinse into the same centrifuge tube; each centrifuge tube was diluted to 50mL with Milli-Q water, capped and mixed well by hand.

In conclusion, the invention has the following beneficial effects: the test method of the present invention can be used to produce reliable and consistent results within the detection and analysis range of elemental impurities As (75), Cd (114), Hg (202) and Pb (208) (10-300% of the working standard concentration) in cannabis extracts and cannabis oil products.

Detailed Description

TABLE 1 limiting values for elemental impurity content in botanical drug and botanical drug preparations

(USP-USP Committee for USP-United states Pharmacopeia standards; NMT-non-invasive micrometering techniques)

The ICP-MS technique is unique in performing elemental analysis:

(1) the high temperature of the plasma effectively eliminates all disturbances of Flame Atomic Absorption (FAA), such as the formation of refractory compounds, suppressors, and the like.

(2) The atomizing elements are determined by their mass to charge ratios, each element being unique. Furthermore, all elemental isotopes are established; therefore, the possibility of finding interference in ICP-ES (ICP emission) is greatly reduced.

(3) The sample is pumped into the interface, ensuring a well-defined sample introduction rate. The typical errors found in the sample introduction rate in the FAA are also reduced. It is no longer dependent on the physical properties (viscosity, density, liquid level) of the sample aspiration process in the FAA.

TABLE 2 verification method

3.0 test method

3.1 reference Standard

All standards used in this study are certified materials suitable for tracking metal analysis.

Table 3: reference standard used in the present verification

Standard article	Suppliers of goods
		Arsenic standard (-1000 mg/L)	Sigma aldrich
Cadmium standard (1000 mg/L)	Sigma aldrich
		Lead standard (1000 mg/L)	Sigma aldrich
Mercury standard (1000 mg/L)	Sigma aldrich
		Bismuth, indium&Germanium internal standard (100ppm)	Agilent

3.2 reagents and solvents

Table 4: reagents and solvents used in this assay

Reagent	Suppliers of goods	Grade
			Concentrated nitric acid	Fisher chemistry	Trace metals
Ultrapure water	Self-made	-
			100% ethanol	Commercial ethanol	Reagent

3.3 sample information

Table 5: sample for validation

3.4 instruments

Verification is carried out in 3 batches

Table 6: apparatus for use

Instrument for measuring the position of a moving object
	Agilent 7900 inductively coupled plasma mass spectrometer
Antopa microwave GO digestion system
	Micro-pipette
Analysis balancing instrument

3.5 Standard, internal Standard, blank and sample preparation

Standards, internal standards, blanks and samples were prepared according to the test methods specified in the attached text

3.6 Instrument settings

Test method according to the attached specification

4.0 verification parameters

4.1 System applicability

System applicability indicates that the analysis system can accurately measure the target value in repeated measurements. The standard solution was freshly prepared each time before analysis. The standard and blank solutions were injected as specified in the attached test methods.

During this method validation, system suitability was performed 5 times. Before and after injection of the samples, the working standard solution was injected and the system float was calculated for each run.

Acceptance criteria:

the correlation coefficient (R) for each target element in the calibration standard curve is NLT 0.99.

Each target element floats no more than 20%

Table 7: system suitability results in first batch

Name (R)	R	Standard calibration float (%)
			As(75)	1.0000	0.1
Cd(114)	1.0000	-2.4
			Hg(202)	1.0000	-5.2
Pb(208)	1.0000	1.9

Table 8: system suitability results in batch 2

Name (R)	R	Standard calibration float (%)
			As(75)	0.9999	5.9
Cd(114)	0.9998	-1.0
			Hg(202)	1.0000	-0.1
Pb(208)	1.0000	0.1

Table 9: system suitability results in batch 3

Name (R)	R	Standard calibration float (%)
			As(75)	0.9999	-1.6
Cd(114)	1.0000	-0.9
			Hg(202)	1.0000	-0.8
Pb(208)	0.9999	1.5

And (4) conclusion:

as shown in tables 7,8 and 9, all system suitability requirements met all acceptance criteria in 4 runs, as shown in tables 7,8 and 9. This demonstrates that the system is reliable for the determination of As (75), Cd (114), Hg (202) and Pb (208).

4.2 limit of quantitation (LOQ) and Limit of detection (LOD)

To determine LOQ and LOD, blank reagents were injected 6 times and the standard deviations of As (75), Cd (114), Hg (202) and Pb (208) were calculated. LOQ and LOD are calculated As specified in the following formulas for As (75), Cd (114), Hg (202) and Pb (208). For a summary of the results, see table 11.

The quantitative limit is equal to 10 times the standard deviation.

Standard deviation of LOQ ═ 10 × blank reagent

The limit of detection is equal to 3 times the standard deviation.

LOD-standard deviation of 3 × blank reagent

Acceptance criteria for quantitative limits:

the calculated LOQ should be below the lowest concentration level obtained in the accuracy study.

Acceptance criteria for detection limits: the calculated LOD should be NMT × 0.1 times the specification limit concentration (table 11).

Table 10: LOD/LOQ in first batch 1

And (4) conclusion:

results from the LOQ and LOD studies were determined to meet all acceptance criteria. The LOQ and LOD for each of As (75), Cd (114), Hg (202) and Pb (208) determined are described in Table 11.

4.3 accuracy

The accuracy of the analysis process represents the closeness of agreement between the traditional true or acceptable reference value and the found value. The accuracy studies were performed at 10%, 50%, 100%, 150% and 300% of the limiting concentrations of As (75), Cd (114), Hg (202) and Pb (208). The accuracy study was performed in two runs.

4.3.1 accuracy: hemp oil

Cannabis oil is a supercritical carbon dioxide extract (extract) dissolved in MCT (medium chain triglycerides). Thus, hemp oil is considered the worst case for MCT. Thus, the method validation results for MCT oil can be inferred from cannabis oil, except for specificity.

Table 11: preparation of intermediate Standard solution 1 (all stock Standard concentrations-1000 mg/L).

Preparation of intermediate standard solution 2:

1.0mL of the intermediate solution 1 was transferred to a 100mL volumetric flask and diluted to volume with diluent and mixed well.

Spiked sample solutions at 10%, 50%, 100%, 150% and 300% levels of spiked hemp oil were prepared in triplicate and corresponding volumes of intermediate standard solution-2 (table 13) were pipetted into separate 50mL volumetric flasks. In addition, 3 additional samples were prepared for precision repeatability testing, which were labeled at 100% level.

Table 12: accuracy benchmarking level

Spiked samples with hemp oil added:

accurately weigh 0.1-0.9g of cannabis oil into a 50mL centrifuge tube (18 samples total).

A quantity of nitric acid, hydrochloric acid, the volume of the intermediate standard-2 solution specified in table 14, 200 μ Ι _ of the internal working standard solution and 50 μ Ι _ of the gold standard were transferred to the same centrifuge tube, the cap tube was loosened and allowed to cool in a fume hood.

Each tube was then capped and shaken by hand and the sample transferred to the digestion tube.

All samples were digested in a multiband GO digestion system (ramp time: 10 min; target temperature: 190 ℃; hold time: 20 min).

After digestion was complete, the samples were transferred to 50mL centrifuge tubes. The digestion tubes were rinsed with ultrapure water and the rinse solutions were combined into the same centrifuge tube. Each tube was diluted to 50-mL mark with ultrapure water, capped and mixed well by hand.

All results are summarized in table 13 below.

Table 13: value of accuracy

And (4) acceptance standard:

the normalized recovery was between 70% and 150% of theoretical for the average of three replicates of each concentration level.

And (4) conclusion:

according to the results of the cannabis oil accuracy study summarized in table 16, all recovery criteria were met. Thus, the test method is accurate for the determination of As (75), Cd (114), Hg (202) and Pb (208) in hemp oil, in the range of 1.2ppb to 36ppb for As (75); for Cd (114) and Pb (208), 0.4ppb to 12 ppb; for Hg (202), 0.8ppb to 24 ppb.

4.3.2 accuracy: cannabis sativa extract

The second test was performed on cannabis extracts. Single unlabeled samples of cannabis extracts were prepared.

The hemp extract spiked sample solutions at 10%, 50%, 100%, 150% and 300% were prepared in triplicate by pipetting the corresponding volumes of intermediate standard solution-2 (table 13) into separate 50mL volumetric flasks. In addition, 3 samples labeled at 100% level were prepared for precision repeatability testing.

Preparation of labeled sample of cannabis extract:

accurately weigh 0.1-0.9g of cannabis extract into 50mL centrifuge tubes (18 samples total).

Add 1mL of ethanol to each tube. Each tube was then capped and vortexed until completely dissolved.

A quantity of nitric acid, hydrochloric acid, the volume of the intermediate standard-2 solution specified in table 12, 200 μ Ι _ of the internal working standard solution and 50 μ Ι _ of the gold standard were transferred to the same centrifuge tube, the cap tube was loosened and allowed to cool in a fume hood.

Each tube was then capped and shaken by hand and the sample transferred to the digestion tube.

All samples were digested in a multiband GO digestion system (ramp time: 10 min; target temperature: 120 ℃; hold time: 15-25 min).

After digestion was complete, the samples were transferred to 50mL centrifuge tubes. The digestion tubes were rinsed with Milli-Q water and the rinses were combined into the same centrifuge tube. Each centrifuge tube was diluted to 50-mL with Milli-Q water, capped and mixed well by hand.

All results are summarized in table 14 below.

Table 14: accurate value

And (4) acceptance standard:

the theoretical value of the mean of the triplicate formulations at each concentration was 70% to 150%.

And (4) conclusion:

based on the cannabis extract accuracy study results summarized in table 14, all recovery criteria were met. Thus, the test method is accurate for the determination of As (75), Cd (114), Hg (202) and Pb (208) in cannabis extracts, in the range of 1.2ppb to 36ppb for As (75); in the range of 0.4ppb to 12ppb for Cd (114) and Pb (208); for Hg (202), in the range of 0.8ppb to 24 ppb.

4.4 range

The calibration range for each metal is within the linear range of the method: test solutions containing residues outside the calibration range level can be diluted to concentrations within the calibration range.

And (4) acceptance standard:

attesting to range by complying with accuracy recovery requirements.

And (4) conclusion:

the accuracy recovery of cannabis oil and cannabis extract meets all requirements, As (75) is 1.2ppb to 36ppb, Cd (114) and Pb (208) are 0.4ppb to 12ppb, Hg (202) is 0.8ppb to 24 ppb.

4.5 precision

4.5.1 repeatability: hemp oil

Table 15: reproducible cannabis oil results summary

And (4) acceptance standard:

the relative standard deviation RSD of each target element is NMT 20% (N ═ 6).

And (4) conclusion:

a summary of the reproducible cannabis oil results of table 15 meets all acceptance criteria. Thus, the test method ARA-18-007.00 protocol was found to be accurate for the determination of As (75), Cd (114), Hg (202) and Pb (208) in hemp oil.

4.5.2 repeatability: cannabis sativa extract

Table 16: summary of reproducible cannabis extract results

4.5.3 intermediate precision

On different days, 6 separate solutions of cannabis oil and 6 separate solutions of cannabis extract samples were prepared, with a standard concentration of 100%, see AR-19-001 pg.01-10. All results are summarized in table 17 cannabis oil and table 18 cannabis extract.

The results of the intermediate precision and repeatability studies were combined for a total of 12 analyses, and the results are shown in table 19.

Table 17: intermediate precision results for hemp oil

Table 18: intermediate accuracy results of cannabis extracts

Table 19: repeatability and intermediate precision combined results

And (4) acceptance standard:

each target element RSD is NMT 20% (N ═ 6)

Each target element RSD is NMT 25% (N ═ 12)

And (4) conclusion:

table 17 summary of intermediate accuracy of cannabis oil, table 18 intermediate accuracy of cannabis extract and table 19 combined results meet all acceptance criteria. Thus, the test methods were found to be accurate and reproducible for the determination of As (75), Cd (114), Hg (202) and Pb (208) in cannabis oil and cannabis extracts.

4.6 specificity

Specificity is demonstrated by adherence to accuracy and precision requirements.

5.0 reference

SOP 020-031.00: drug and drug assay validation

ICHQ2 (R1): verification of the analysis program: text and method

USP-41<232> elemental impurity-limiting amount

USP-41<233> elemental impurities-procedure

Ph, eur, 2.2.58 "inductively coupled plasma-mass spectrometry"

Ph. eur.2.2.23 "atomic absorption spectroscopy" — method I.

Incumbent ph.eur.2.4.27. Heavy metals in herbal and herbal preparations "

USP-41<561> "article of plant origin"

"operating Agilent 7900ICP-MS Using Mass Hunter 4.1 software"

"operation of Multiband-Go microwave digestion System"

6.0 conclusion

All acceptance criteria were met and "method for determination of elemental impurities (arsenic, cadmium, lead and mercury) in MCT oil, cannabis extract and cannabis oil products by ICP-MS" was validated at kanabis pharmaceutical limited.

The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present invention.

Claims (10)

1. A method for detecting element impurities in a hemp extract and a hemp oil product is characterized by comprising the following specific steps:

(1) selecting an analysis method: mass spectrometric detection using inductively coupled plasma

(2) Determination of the reference standard: selecting standard arsenic, cadmium, lead and mercury products with concentration of less than 1000 mg/L;

(3) selecting a reagent solvent: selecting concentrated nitric acid, ultrapure water and 100% ethanol; wherein the mass concentration of the concentrated nitric acid is 68-97.5%;

(4) selecting a sample: selecting cannabis extract and cannabis oil;

(5) preparing standards, internal standards, blanks and samples;

(6) the test was performed according to the following verification parameters:

the system applicability is as follows;

b, quantitative limit value and detection limit value;

c, testing accuracy: comprising i) testing the accuracy of cannabis oil;

ii) accuracy testing of cannabis extracts;

d, testing the range;

e, testing the accuracy: comprising i) a reproducibility test for cannabis oil;

ii) reproducibility testing of the cannabis extracts;

iii) intermediate precision testing;

and F, testing the specificity.

2. The method of claim 1, wherein the quantification limit is equal to 8-12 times the standard deviation of the blank reagent.

3. A method of detecting elemental impurities in cannabis extracts and cannabis oil products as claimed in claim 1, wherein the calculated limit is below the lowest concentration level obtained in the accuracy study.

4. A method of detecting elemental impurities in cannabis extracts and cannabis oil products as claimed in claim 2, wherein the limit of detection is equal to 2-4 times the standard deviation of the blank reagent.

5. A method of detecting elemental impurities in cannabis extracts and cannabis oil products as claimed in claim 3, wherein the detection limit is equal to the non-invasive micro-measurement technique multiplied by a 0.05-0.15 time specification quantitation limit concentration.

6. A method of detecting elemental impurities in cannabis extracts and cannabis oil products as claimed in claim 1, the accuracy test being used to test the proximity of the amount found to a known amount added; triplicates were prepared at 50%, 100%, 150% of the limit; the average recovery per grade was 70-150%.

7. A method of detecting elemental impurities in cannabis extracts and cannabis oil products as claimed in claim 1, wherein the accuracy test is performed in more than 6 replicates at the 100% limit.

8. The method of claim 1, wherein standard calibration float range for system applicability is-5.2% -5.9%.

9. A method of detecting elemental impurities in cannabis extracts and cannabis oil products As claimed in claim 1, the accuracy of the elemental impurity As (75) is in the range of 1.2ppb to 36 ppb; accuracy of Cd (114) and Pb (208) is 0.4ppb to 12 ppb; the accuracy of Hg (202) is 0.8ppb to 24 ppb.

10. A method of detecting elemental impurities in cannabis extracts and cannabis oil products as claimed in claim 1, the method of preparing a spiked sample of cannabis extract comprising the steps of:

1) accurately weighing 0.1-0.3g of cannabis sativa extract into a 50mL centrifuge tube;

2) adding 0.5-3mL of ethanol to each tube; each tube was then capped and vortexed until completely dissolved;

3) transferring nitric acid, hydrochloric acid, intermediate standard-2 solution, 150-;

4) then, tightly covering each tube, uniformly shaking by hand, and transferring the sample into a digestion tube;

5) digesting all samples in a multi-band GO digestion system; digestion time: 5-15 minutes; target temperature: 120 ℃ to 210 ℃; retention time: 15-25 minutes;

6) after digestion, transferring the sample into a centrifuge tube; rinsing the digestion tube with ultrapure water, and combining rinsing liquids into the same centrifuge tube; each tube was diluted to 30-100mL mark with ultrapure water, capped and mixed well by hand.