There are many reasons why you get a variation in results between labs…and not that many of those reasons have to do with the actual instrument.

We will put the reasons into 3 categories:

1. Instrument design

2. Sample preparation

3. Test method design & reporting

Let`s go over them in more detail.

1. Instrument design

HPLCs are in general reliable instruments and in many ways rather simple in design. There are a few things that can influence the results – most of them are very minor and the actual instrument is very rarely the cause of these discrepancies.

Pump – pumping flow rate issues can cause unstable baseline and variation in peak shape causing the software to incorrectly establish the baseline and the peak height or surface area. In the case of binary/quaternary pumps, the mixing valves and mixers can cause issues with gradients resulting in misshaped peaks.

Detector – depending on detector setting, there are scenarios where the detector would cause the result to be off. Detector measures signal in series as data points. If the scanning frequency is set to be too low, then in cases where compound elutes from the column very quickly demonstrated as a very sharp peak, the detector set to scanning every second may miss the top of the peak as within that second the top of the peak would be cut off due to slow detector reading.

Injector – there may be issues with the injector or injection technique itself. The injector may leak resulting in not all the sample being injected. Likewise, if the analyst injects the sample with a bubble or does not puncture the needle seal inside the injector causing the sample to go around the needle rather than inside the injector, then the measured result would be lower because less sample was injected.

Sample loop – most HPLCs use sample loops to assure the same volume of sample is measured. We use a 20 microlitre sample loop. This being said, these loops are made from stainless steel with a typical internal diameter of 0.015 – 0.020″. According to Rheodyne (a leading manufacturer of sample loops), the internal volume on 20uL loops is +/-10% to 20%. This means these loops are not very interchangeable between instruments without optimizing the testing methods. Furthermore, if the analyst does not inject at least 3 to 5 times the volume of the sample loop, it may not get completely filled causing discrepancies.

HPLC column – wrong choice of column and testing method design have a profound impact on the measurement accuracy – we will discuss in more detail later. Columns are consumables with their life expectancy and need to be treated that way. Old columns, or wrong columns cause misshaped peaks resulting in wrong results.

Autosampler – if not optimized and injection volume not checked regularly, they may give inaccurate measurements.

How do I know if I have an inaccurate instrument?

When you measure commercial standards, if they are off, it is most likely due to instrument set up; although the testing method can have an impact as well – more on this later.

2. Sample preparation

Sample preparation tends to have the highest impact on result variation between labs. Each sample preparation must meet 2 criteria:

a) sample must be representative – i.e. no matter which portion of the sample you take, you get the same result. In flower and biomass samples, this is achieved by drying the sample and evenly distributing it by cutting, milling, or grinding. Because the cannabinoids are stored in trichomes, they are not equally distributed across the flower – you will find that in the top portion of the bud there are fewer measured cannabinoids than in the middle of the bud due to the density of cannabinoid-bearing trichomes. Therefore it is important to create a representative sample from the bud. Equally, this applies to when you are testing your crop and using two different flowers for the analysis.

Similar idea is when testing oils, extracts, or tinctures. Some are separated into two layers inside the bottle with one layer containing more of certain cannabinoids than the other, for instance, due to different lipophilicity/attraction of some cannabinoids vs. others. This can happen in extracts when distillation is not completed, or the final product is incorrectly formulated and “falls apart” due to storage conditions, temperature, etc. It is a good habit to vigorously shake the bottle prior to sampling for analysis. As mentioned before regarding batch consistency, the same applies to liquid samples. One bottle from the manufacturer may not be the same as the bottle right next to it. One can be filled from the top of the manufacturing batch that may be lighter, while the other can be filled with the heavier bottom of the manufacturing batch – this is all manufacturer`s quality control issue and can`t be solved by making a representative sample 

b) cannabinoids must be fully dissolved in the extraction solvent. Some labs use crazy techniques to extract the cannabinoids including heating and sonicating for an hour. Our extraction techniques are simple and proven. While flowers tend to be straightforward, edibles and some extracts pose a challenge. There is a common myth across the industry that measuring cannabinoids in chocolate is inaccurate. All the studies we have seen are simply transposing extraction techniques from flowers – for instance, all the studies we have seen use methanol, which doesn`t make sense as chocolate is made from grease/butter and methanol has a hard time penetrating it to extract cannabinoids.

Our sampling protocols are easy to follow, tested, and proven to work. We continue refining the protocols based on customer feedback.

3. Test method design and reporting

There is a common misconception that purchasing commercial standards and testing them using a method published in literature gives a valid method. It takes a very long time and a large number of tests to assess and validate the analytical method on real samples. Various types of samples have different types of matrices with interferences on the measurement. Even the standards themselves need to be compared from several sources to assure their accuracy. We have extensive experience in testing real-life samples and we use this experience to optimize our methods.

The way how the measured data is processed can have a significant impact on the accuracy of the results, especially at lower concentrations towards the lower end of calibration curves, where results can be 40%-50% off if not processed properly.

More on the topic of calibration curves and their impact on results can be found here.

Coming soon.

Below are several best practices to follow in order to improve the accuracy of weighing samples.

1. Make sure your electronic balance is on a stable, flat surface and is level. Check the bull’s eye leveling circle at the back to confirm the bubble is in the center. If not, level the balance using the balance feet.

2. Make sure you turn the balance on at least 30 minutes before doing measurements. It takes ca. 30 minutes for the balance to warm up.

3. Close the glass enclosure and tare the balance before weighing.

4. Use gloves to hold the vial to prevent greasy residues from your fingers to influence the weight of the vial.

5. When weighing a vial or sample, close the glass enclosure to prevent the draft to impact the measurement. Always wait 5 seconds for the weight to stabilize.

6. If you spill or leave any residues inside the balance, clean it right away. Use a small brush (toothbrush) to remove solids and powders from the balance. Make sure solids do not get under the weighing pan inside the balance weighing mechanism.

Coming soon.

There are many reasons why you get a variation in results between labs…and not that many of those reasons have to do with the actual instrument.

We will put the reasons into 3 categories:

1. Instrument design

2. Sample preparation

3. Test method design & reporting

Let`s go over them in more detail.

1. Instrument design

HPLCs are in general reliable instruments and in many ways rather simple in design. There are a few things that can influence the results – most of them are very minor and the actual instrument is very rarely the cause of these discrepancies.

Pump – pumping flow rate issues can cause unstable baseline and variation in peak shape causing the software to incorrectly establish the baseline and the peak height or surface area. In the case of binary/quaternary pumps, the mixing valves and mixers can cause issues with gradients resulting in misshaped peaks.

Detector – depending on detector setting, there are scenarios where the detector would cause the result to be off. Detector measures signal in series as data points. If the scanning frequency is set to be too low, then in cases where compound elutes from the column very quickly demonstrated as a very sharp peak, the detector set to scanning every second may miss the top of the peak as within that second the top of the peak would be cut off due to slow detector reading.

Injector – there may be issues with the injector or injection technique itself. The injector may leak resulting in not all the sample being injected. Likewise, if the analyst injects the sample with a bubble or does not puncture the needle seal inside the injector causing the sample to go around the needle rather than inside the injector, then the measured result would be lower because less sample was injected.

Sample loop – most HPLCs use sample loops to assure the same volume of sample is measured. We use a 20 microlitre sample loop. This being said, these loops are made from stainless steel with a typical internal diameter of 0.015 – 0.020″. According to Rheodyne (a leading manufacturer of sample loops), the internal volume on 20uL loops is +/-10% to 20%. This means these loops are not very interchangeable between instruments without optimizing the testing methods. Furthermore, if the analyst does not inject at least 3 to 5 times the volume of the sample loop, it may not get completely filled causing discrepancies.

HPLC column – wrong choice of column and testing method design have a profound impact on the measurement accuracy – we will discuss in more detail later. Columns are consumables with their life expectancy and need to be treated that way. Old columns, or wrong columns cause misshaped peaks resulting in wrong results.

Autosampler – if not optimized and injection volume not checked regularly, they may give inaccurate measurements.

How do I know if I have an inaccurate instrument?

When you measure commercial standards, if they are off, it is most likely due to instrument set up; although the testing method can have an impact as well – more on this later.

2. Sample preparation

Sample preparation tends to have the highest impact on result variation between labs. Each sample preparation must meet 2 criteria:

a) sample must be representative – i.e. no matter which portion of the sample you take, you get the same result. In flower and biomass samples, this is achieved by drying the sample and evenly distributing it by cutting, milling, or grinding. Because the cannabinoids are stored in trichomes, they are not equally distributed across the flower – you will find that in the top portion of the bud there are fewer measured cannabinoids than in the middle of the bud due to the density of cannabinoid-bearing trichomes. Therefore it is important to create a representative sample from the bud. Equally, this applies to when you are testing your crop and using two different flowers for the analysis.

Similar idea is when testing oils, extracts, or tinctures. Some are separated into two layers inside the bottle with one layer containing more of certain cannabinoids than the other, for instance, due to different lipophilicity/attraction of some cannabinoids vs. others. This can happen in extracts when distillation is not completed, or the final product is incorrectly formulated and “falls apart” due to storage conditions, temperature, etc. It is a good habit to vigorously shake the bottle prior to sampling for analysis. As mentioned before regarding batch consistency, the same applies to liquid samples. One bottle from the manufacturer may not be the same as the bottle right next to it. One can be filled from the top of the manufacturing batch that may be lighter, while the other can be filled with the heavier bottom of the manufacturing batch – this is all manufacturer`s quality control issue and can`t be solved by making a representative sample 

b) cannabinoids must be fully dissolved in the extraction solvent. Some labs use crazy techniques to extract the cannabinoids including heating and sonicating for an hour. Our extraction techniques are simple and proven. While flowers tend to be straightforward, edibles and some extracts pose a challenge. There is a common myth across the industry that measuring cannabinoids in chocolate is inaccurate. All the studies we have seen are simply transposing extraction techniques from flowers – for instance, all the studies we have seen use methanol, which doesn`t make sense as chocolate is made from grease/butter and methanol has a hard time penetrating it to extract cannabinoids.

Our sampling protocols are easy to follow, tested, and proven to work. We continue refining the protocols based on customer feedback.

3. Test method design and reporting

There is a common misconception that purchasing commercial standards and testing them using a method published in literature gives a valid method. It takes a very long time and a large number of tests to assess and validate the analytical method on real samples. Various types of samples have different types of matrices with interferences on the measurement. Even the standards themselves need to be compared from several sources to assure their accuracy. We have extensive experience in testing real-life samples and we use this experience to optimize our methods.

The way how the measured data is processed can have a significant impact on the accuracy of the results, especially at lower concentrations towards the lower end of calibration curves, where results can be 40%-50% off if not processed properly.

Yes. Very much so! Many of our customers are commercial testing labs.

Your accuracy of measurement is not dependent on the HPLC instrument as much. There are some nuances, which you can review in this article: https://ctinstruments.freshdesk.com/support/solutions/articles/69000799047-why-are-there-differences-in-results-between-labs-

As a testing lab, you will need to have quality control and instrumentation validation processes to test independent commercial calibration standards; in other words, you will be testing independently purchased commercial standards just as any other lab to confirm the instrument is operating correctly and you will be recording it for auditing purposes. Our software allows you to do so and we have the processes set up to do quality control, validation, and audit. In other words, you will measure, for instance, THC commercial standard with concentration of 100ug/mL and if you get measured concentration of say 101.2 ug/mL, you can conclude that the instrument is operating correctly and you are able to reliably measure THC concentrations.

So basically, you will be doing the same things as labs regardless of whether you have a license or not – all our customers (even those who are not labs) do this validation with commercial standards as our software allows for the quality control and generation of audit trail. None of our customers had issues with passing of the standards so far and the HPLCs are operating as expected.

Here is more on the reproducibility of the results with our instrument: https://ctinstruments.freshdesk.com/support/solutions/folders/69000583159

Below you can find our certificate from the National Institute of Standards and Technology in USA that confirms our results in comparison to ca. other 150 commercial labs in North America. If you go to pages 2-4, you will see we are where all the other labs with all our results; none of these labs used our instrument, all were expensive HPLCs from Agilent, Shimadzu, etc.

https://cannabistestingsimplified.com/ctinstruments-cannabis-hplc-analyzer-comparison-to-other-labs/

You can also review the comparison of our results to labels on purchased products on our website under Test Results or Application notes – they have always been pretty much identical to what is on the label on all products that we tested.

If you follow the testing protocols and do the quality control and validation, the accuracy is no different as our instrument operates the same way as HPLCs from all the other manufacturers. You would have to do the same quality controls and commercial standards validations on those instruments as well.

You can use any commercial HPLC column. Our columns are standard C18, 150×4.6mm with 5um particles. You can use columns with different specifications, such as 150×4.6mm with 2.7um particles – this will double the pressure if the same flow rate is used. Our HPLC is rated to 6,300 psi (note that most commercial columns are rated to 6,000 psi; unless designed for UHPLC system). You can use other types of columns than C18, such as C8, or normal phase. Our HPLC operates the same way as other HPLCs, with some limitations.

We recommend using our columns as with columns from other manufacturers, you would have to re-do all the calibration curves and standardize the processes for a different column. Every manufacturer uses a different type of packing material (although still C18, they have different terminal chemistries that interact with cannabinoids in a different way, changing retention times and signal response). Developing these from scratch costs thousands of dollars and months of work as besides figuring out the correct conditions (while sometimes reported in the literature), you need to test many real-life samples to find potential negative interactions and problems. Unfortunately, developing a robust, validated method is not as simple as creating calibration curves from commercial standards. By purchasing our system and sticking to our columns, you have everything already developed and validated.

Additionally, our columns cost the same as columns from other manufacturers. We test each column individually with cannabinoids before shipping to confirm the accuracy and retention times. Even the same columns have variations in retention times due to the packing process.

Yes. We supply a guard column as a standard solution included in the package.

Our HPLC method test run is 13.2 minutes. You need ca. 35 minutes to start up the HPLC and condition the column at the beginning of the day and 40 minutes to clean the column at the end of the day. In between, you can inject continuously until you run out of samples. We have an autosampler with 45 samples capacity so HPLC would run those until all finished (or as programmed).

Assuming 4 samples per hour and 8-hour shift, you can do ca. 20-24 samples per day, depending on how you organize the receiving and extraction process. With the use of autosampler, you can leave the testing unattended.

Yes, it can be used to analyze many other substances – anything that can be measured with an HPLC using isocratic mode – there are hundreds of isocratic tests in environmental, food, lubricants, and other industries. The only limitation is using an isocratic method and the use of a UV detector. Most compounds have isocratic methods already developed and published, or readily available. We continue adding tests, such as terpenes, pesticides, and more cannabinoids, beyond the current 11.

We use a single wavelength detector with a spectral range 180 to 680 nm. You can set up any wavelength in that range and do measurements at that wavelength. All cannabinoids are measured at 220nm regardless of the type of UV detector. While PDA/DAD detectors can scan multiple wavelengths at the same time, those having HPLCs with PDA/DAD detectors measuring cannabinoids still do it at a single wavelength of 220nm.

With our HPLC, if you are interested in a UV spectrum of a specific compound, you can do a spectral scan from 180 to 680 nm. The detector will run through the wavelengths in 1nm increments and will plot the UV spectrum of the substance. After that, you can set the detector to the desired wavelength (ideally the one with the highest signal strength) and then do analyses at that specific wavelength.

We use a deuterium lamp from Hamamatsu Photonics and the lamp is manufactured in Japan. It has a half-life of 2,000 hours.

Yes, you can connect other detectors, such as fluorescence or electrochemical detector.

No. Our HPLC has a reciprocating pump that is not capable of mixing solvents, hence isocratic methods only can be used.

This being said, you can program your own isocratic method.

Yes. You can adjust the temperature, wavelength, flow rate, method length, and program changes/combination of these during the run.

We use a reciprocating pump that is not capable of mixing solvents, hence only use isocratic methods. This being said, most technical analyses have methods developed both in gradient and isocratic modes.

You can use any HPLC-grade solvent (water, acetonitrile, methanol) as long as they meet the below requirements:

1. Brand – it doesn`t matter. The most common brands are: VWR, JTBaker, TCI, Supelco, Honeywell, B&J

2. Gradient vs. isocratic – it doesn`t matter. Purchase the most cost-effective option.

3. Important specification: Absorbance at 220nm should be low (max. around 0.3)

As long as the solvent is HPLC-grade and has low absorbance in the 200nm to 220nm region, then you can buy the most cost-effective solvent.

For the customers who are not supplied HPLC solvents as part of their starter kit, we recommend buying 1L bottles first with larger volumes subsequently. You can use the 1L bottles to prepare the mobile phase or extract cannabinoids. These smaller bottles are easier to work with and can easily be filled from larger bottles later.

Once you have paid for your purchase, your order will be confirmed. Should you not request cancellation within 48 hours of the time your order was confirmed (the “Period”), your order will become firm and your payment will become non-refundable. Should you cancel your order within the Period, the purchase price will be refunded minus the bank transaction fees.

Reproducibility is a measure of how repeatable results are. There are several factors that have an impact on the reproducibility of results.

On a high level, there is:

A. reproducibility of the instrument measurement, which is influenced by the instrument design

B. reproducibility of the testing method, which is influenced by the testing method design

Reproducibility of results is influenced by ow repeatable:

1. your sample preparation is (hence it is important to always use a uniform representative sample),

2. your extraction and dilution solvent measurements are

3. your extraction times are

4. your injection technique is – therefore it is important to avoid having any bubbles in the injection syringe and always inject at a minimum 3x20uL (sample loop volume)

5. mobile phase is – pH, composition

6. instrument settings are – flow rates, temperatures

7. instrument itself – pump, column, detector

In the context of reproducibility, we will be considering everything constant and will consider only the instrument by injecting the same sample several times to judge the reproducibility.

Reproducibility is expressed as a relative standard deviation (RSD), which is a measure of variation and distribution of numbers, in our case results obtained.

In the world of analytical chemistry, RSD is highly dependent on where the measure is done – at the low end of the signal, middle, or the high end. High end (upper limit of calibration curve) and the middle tend to be more accurate with typical RSD 5% – 10%, while at the low end (lower limit of detection), the common RSDs are 10% – 20%.

Below are the results of measuring a typical hemp sample, followed by an image of stacked 3 consecutive measurements. There are two observations from this measurement:

1. The retention times are consistent and repeatable, giving the confidence in the ability to correctly identify the correct cannabinoids. Retention times relative standard deviation (RSD) range from 0.27% to 0.69%.

2. The measured concentrations are very repeatable as indicated by the height/area of each peak. The calculated relative standard deviation (RSD) in this case ranges from 1.19% to 1.70%, while for cannabinoids with very low concentrations manifested as very small peaks (in this sample CBN, d9-THC, and d9-THCA) the calculated RSD ranges from 4.62% to 5.63%. In case of need to further refine the latter 3 compounds and improve both accuracy and RSD, the sample would simply be less diluted resulting in higher peaks for these 3 compounds.

Below are results of consecutive measurements of cannabinoids commercial standard at 5% mark of the calibration curve.

As seen in the image, the relative standard deviation (RSD) is ranging from 0.37% to 1.23% for these cannabinoids. We can also observe RSD of retention times ranging from 0.22% to 0.27%.

Typically, we try to fill the order within 2 weeks from the payment receipt. We ship by UPS and it takes 1-2 weeks to deliver. We have had instances where the package was inspected by the government authorities causing a delay of up to 3 weeks.

In some instances, we do not ship the HPLC solvents to certain countries due to local dangerous goods regulations. In such case, we will not quote HPLC solvents for you and will ask you to purchase them locally. We ship HPLC solvents as dangerous goods by DHL. Due to DG regulations, we are able to ship only a maximum of 1L of each HPLC solvent.

We quote the most economic shipping cost using a courier, UPS in most cases. The shipment typically consists of 5 packages, two of which are heavy and bulky. One of the packages is dangerous goods and the shipment cost includes dangerous goods shipment processing typically charged at $125 by the courier on top of the shipping cost. In addition, the shipping quote includes the shipment of commercial standards that are shipped by Fedex Overnight Priority cold chain usually at around US$150-$200 to most of our customers. Lastly, the shipping quote includes insurance. Considering all this, we try to keep the shipping costs as low as we can.

Below is an image of a typical shipment excluding the standards.

We arrange the shipment of commercial standards purchased from Absolute Standards, a standards manufacturer based in Hamden, CT, USA.

We arrange the shipment only after you receive all your HPLC packages.

Commercial standards are shipped by Fedex overnight priority. The package is shipped via cold chain with samples kept on ice and it costs ca. USD 150-200 in shipping costs, hence it is important to receive the standards at the earliest and store in a freezer.

Commercial standards need to be stored in a freezer. This being said, they are shipped in sealed ampules and are stable until open. If they get stored at room temperature in a warehouse over the weekend, they will still be fine as they are sealed. We try to coordinate the shipments to avoid interruptions or weekends.

The warranty is one year from the date of purchase except for electronics (90 days) and consumables, such as UV lamps and HPLC columns (90 days). Canadian purchasers, subject to restocking charges, can return their purchase within 14 days of the purchase date. Non-Canadian purchasers cannot return their purchases. Items received by the Company or Retailer after the specified 14 days, for whatever reason, will not be accepted for return. Contact the Company for a Returned Merchandise Authorization (“RMA”). All returns must be in original, undamaged condition, clean and free from any material and/or residue, and delivered back, at the buyer’s expense, to the place of purchase. There is a 20% restocking fee on all returns plus any applicable additional fees (Banking Transfer Fees). If damaged for whatever reason, in any way, a minimum 25% restocking fee will be applied. Please insure your return against shipping damage, and be sure to include the RMA number. Shipping charges, fees, taxes, duties, etc. are not refundable. Once your merchandise is received and accepted back, a refund of your original purchase price minus the applicable restocking fee and banking charges will be processed and a credit will be applied to the original method of payment. Additional time may be taken by the bank to post the credit to your account.