Cannabis, Cannabinoid (Hemp, THC, CBD, CBN, Marijuana) HPLC Analysis and Testing, Areas for Improvement [*Updated 8/2021]

Over the past few years we have observed an exponential growth in the number of state-level, legal businesses (in the USA) who offer Cannabis Analysis (e.g. Potency Analysis or Profiles) and/or related businesses such as Hemp Oil Extraction. Most related products which incorporate Marijuana, CBD, THC, Cannabinoids, Terpenes and/or other related compounds require formal analytical laboratory testing which should follow good laboratory practices. This article is targeted to help many of the people involved in this new analysis business (or anyone using chromatography as the analytical technique of choice for the same goals).

As a professional chromatography consultant, I have seen a large increase in the number of requests for my services to this new market. Most of these new businesses have questions about obtaining professional training, correct analysis procedures, improving reproducibility, documentation, optimizing method development, how to receive professional training in maintenance of the HPLC system(s) and need hands-on help to optimize the procedures used. Many users are not achieving acceptable results and need help finding out why. They want to know where they can take a class to learn HPLC method development and how to perform the required tests. 

These new businesses would benefit greatly from professional guidance EARLY in their setup and establishment, to improve the internal methods and procedures of analysis used. Time spent on the "front-end" of any process is always time spent wisely (in this context, knowledge and practical experience = confidence). A chromatography professional can quickly identify areas which may need improvement and/or suggest changes that can directly improve your company's accuracy, reproducibility, increase efficiency and of course, impact your bottom-line too. Focus is placed on the exact areas that will benefit you (rather than wasting time with non-targeted approaches, sales biased classes and trial-and-error approaches). 

• Please note that there are NO SCIENTIFICALLY LEGITIMATE ONE DAY, ONE WEEK or ONE MONTH LONG TRAINING CLASSES THAT CAN TEACH YOU HOW TO PERFORM HPLC METHOD DEVELOPMENT or ANALYSIS. NONE AT ALL. Most types of "Certification" offered are completely without scientific merit or value. The training needed takes many years of hands-on experience, in an industrial laboratory (not a school), to acquire just a basic level of proficiency (*Emphasis on 'Basic", not intermediate or expert). Be very cautious of anyone who claims to be able to provide you with all the training you need in a short time period.
  

Generating accurate and reproducible analytical data, esp. with HPLC, SFC or GC requires a great deal of knowledge, formal training and practical hands-on experience (not something which is taught at most university or school programs). These complex techniques require years of bench time and professional hands-on experience to learn). Shipping or selling products which contain unacceptable levels of impurities or which do not meet basic testing or regulatory qualifications could pose a health and/or liability risk. Hire people who have the needed training from industry before setting up the laboratory.

It has been my professional experience that some of the most common training areas that client's would benefit from are: GLP (Good Laboratory Practices/Procedures and SOPs) and additional instrument operational training to demonstrate proficiency in analytical chromatography. Address these areas early on and continuously update them to reduce errors and improve results. Training should continue on a regular basis to gain experience.
 
While each confidential consultation visit I have with a client may show different key issues which need to be addressed, many labs can start to improve their analytical results by addressing and improving how they address:

1. Documentation: Laboratory methods and sample analysis must be conducted using clearly written documentation. This should initially include having Standard Operating Procedures (SOPs) in place for all methods, procedures, qualification, verification and tests used. They should include SOPs, Document Control and Policy documents which also address: Training, Calibration, Maintenance, Frequency of the same, Mobile phase preparation, pH measurement, use of the balance and so on... I find that it is best to create an initial SOP Template to insure document uniformity (i.e. include such fields as: Business Name, SOP #, review/approval date(s)/names, Rev #, materials & tools needed, purpose, procedure steps, pass/fail definitions... plus any needed supporting documents).
2. Sample Preparation Methods: Be sure to document, test, review / standardize specific sample preparation methods, for each sample type. Variations in: temperature, extraction solvent or the solution(s) dissolved in, homogenization or grinding methods, mixing, times used, glass or plastic containers used may result in significant variation of the final reported results.
3. Correct Poor Reproducibility and/or Baseline Instability Issues: In chromatography analysis, if the method(s) used are not stable and reproducible, every time they are run, then little to no scientific value can be obtained from them. Methods used must follow basic good chromatography fundamentals and meet basic guidelines. Baseline noise or instability may directly impact integration results (which directly impacts reported results). Instrumentation must be operated in clean, climate controlled rooms. Failure to reproduce a result within acceptable limits (these will vary per method type) will invalidate the method used. Make sure that SOP's are followed, mobile phase solutions are made fresh each day (do not pre-mix solutions with acids and let them sit for several days before use; do not "top off" bottles), solutions should be degassed, HPLC columns are properly washed and re-equilibrated before each analysis, instruments are maintained (per a SOP) and serviced on a regular basis. 
4. Develop HPLC methods that follow good chromatography fundamentals: Retain, separate and resolve ALL peaks. Insure peak K primes are 2.0 or higher. If you have co-eluted peaks in your method, then method development is not finished. If you have ghost peaks or changing retention times, then you need to stop running samples and find out why. Be careful whose method(s) you use. A method that is "Validated" may not be scientifically valid method to use. Have the method checked by an experienced chromatographer.
   
5. Continuous Training is Required to become Proficient: To be proficient, at a basic level in chromatography, takes most chromatographers several years working in an industrial environment to gain practical hands-on time. That assumes that they have had professional training outside of college, in an industrial lab, and can demonstrate an understanding of the fundamentals of good chromatography. Note, that method development skills require a much higher level of understanding and hands-on training to acquire the needed skill set. Make sure your scientists have the needed level of training to operate, run analysis methods and troubleshoot any issues that come up (and issues will come-up, even under ideal conditions). Please do not make the mistake of thinking they will "figure it out" on their own. Hire people who already have several years of industrial chromatography experience, then provide them with additional training opportunities to advance their skills in the application areas that your business needs.  Get them help NOW, you will save money and time, plus get back on track moving forward with your project.
   

If you want to surpass your competitors and provide clients with the most accurate data, then investing in your employees professional knowledge and hands-on technical training is the fastest route to do so. This is an experience based technique where decades of practical knowledge are needed to improve your skill set. A professional can quickly provide you with practical information and show you techniques that will help you move forward. 1-2 days of on-site training often translates to nearly one years worth of knowledge. What is one-years worth of lost time worth to you?
  
Additional Resources:

• Modern HPLC Method Development Tips (PART I): 
• Modern HPLC Method Development Tips (PART II):
• EXPERT CANNABINOID HPLC Training and CONSULTING SERVICES: 

A Case of Changing Solution pH. Formic Acid Stability in Solution (Methanol)

Real life examples help to better illustrate problems that I am called in to troubleshoot for clients. As a professional scientific consultant, many of my clients have spent months (sometimes years) trying to solve an analytical problem on their own before I am brought in to make the diagnosis and propose a solution. Many years of working in a wide range of scientific fields allows me to identify problems quickly and efficiently saving clients the most money and allowing them to resume work on their projects.

This was the case during a recent consult for a major cannabis testing laboratory. They were having a great deal of difficulty obtaining reproducible results for their analytical testing screens (14 compounds in their analysis with a need for repeatable and accurate results). Variations from 25% to 50% were observed run-to-run over the course of seven days. They assured me they were doing everything in the same way. To begin the troubleshooting process, we started by looking at the actual data gathered and the actual method(s) used to acquire the data. These were evaluated to see if they followed good practices and techniques, also to make sure they had SOP's in place which were clear. Good SOP's must include enough detail to allow anyone reviewing them to prepare samples, standards and/or solutions in the exact same way. Additionally, the HPLC instrumentation was checked and tested to verify it was performing as designed.

After reviewing their training and methodologies on-site, a number of areas of concern were quickly identified. One of the most likely reasons for the variation in values over time was found to be caused by a common mistake in the preparation of mobile phase solutions for the HPLC system. To save time, the client's scientists prepared all organic solvent solutions in advance (~ one month or more), then filtered and stored them at room temperature. For example, their solutions of 0.1% formic acid in HPLC grade Methanol were pre-mixed and stored in glass one liter bottles. These bottles were then put aside, for an average of one month before use. This finding proved key as someone with proper HPLC training would be aware of a well known problem when formic acid is left in pure organic solvent, especially methanol, over time (less so with ACN). Briefly, the formic acid content degrades quickly over time and is often found to be only half of what it was initially after just three or four days (If you have not done so already, this is a simple and useful experiment to run in your lab, monitoring the acid level by titration, not with a pH meter, over time at room temperature in methanol)! This degradation continues over time reducing the amount of acid in solution. If the acid is added to the solution to enhance ionization (i.e. LC-MS; LC-MS/MS) or provide acidification to maintain the sample in a fully ionized form, then as the level of acidification decreases, so does the solution's ability to maintain it. In other words, your HPLC method may change over time (resulting in an in-valid method).

•  I have always promoted the importance of making and using freshly prepared mobile phase solutions (daily), especially where any aqueous solutions are used (to prevent degradation of additives and/or bacterial or fungi growth). However, this precaution does not normally apply to many pure organic solvents, but there are a few very important exceptions to this, formic acid and methanol in this example. 

Changes were made to their SOP's to insure that future solutions of formic acid in methanol were not prepared in advance, but instead, fresh on the day needed only. This coupled with a few basic improvements to their column washing, equilibration and overall training resulted in %RSD of only 0.3% for future analysis runs.

 
As a side note, I have been asked why solutions of formic acid in methanol are sold commercially for HPLC use? I have no answer to this, but respectfully remind everyone that just because something is offered for sale, does not mean it should be purchased. Ask yourself if the item is appropriate for your application? It may not be suitable for your use or application. 

BTW: Please be sure to flush your HPLC system of all organic acids (e.g. acetic, formic) after use and do not leave them in the HPLC system overnight. Even 1% levels of organic acids may be corrosive to stainless steel. 

Common pKa Values for ACIDS & BASES used in HPLC and LC/MS Method Development

pKa (25°C)                              ACID

0.3                                           Trifluoroacetic acid

2.15                                          Phosphoric acid (pK#1)

3.13                                          Citric acid (pK#1)

3.75                                          Formic acid

4.76                                          Acetic acid

4.76                                          Citric acid (pK#2)

4.86                                          Propionic acid

6.35                                          Carbonic acid (pK#1)

6.40                                          Citric acid (pK#3)

7.20                                          Phosphoric acid (pK#2)

8.06                                          Tris

9.23                                          Boric acid

9.25                                          Ammonia

9.78                                          Glycine (pK#2)

10.33                                        Carbonic acid (pK#2)

10.72                                        Triethylamine

11.27                                        Pyrrolidine

12.33                                        Phosphoric acid (pK#3)

Notes: (1) This is a general list of commonly used acids & bases for chromatography applications and not meant to be a comprehensive list of all values. (2) TFA is an overused and very strong acid for many chromatography applications. It also has strong ion pairing properties and can result in high UV noise, vacuum degasser and/or MS contamination. If you must use it, try and use the lowest concentration which results in the desired pH. Example: 0.1 % TFA ~ pH 2.0, 0.02% TFA ~ pH 2.7. (3) Formic acid is a popular alternative to TFA for many applications, esp LC/MS. (4) Not all acids/bases provide "buffering" on their own.

Reference: CRC Handbook of Chemistry & Physics.

Popular LC/MS and HPLC Volatile Mobile Phase Modifiers

For applications which utilize an Evaporative Light Scattering Detector (E.L.S.D.), Charged Aerosol Detector (CAD) and/or Mass Spectrometer Detector with Electrospray Ionization source (e.g. LC/MS, MSD or LC/MS/MS), a fully volatile buffering system is usually required. Many of the common HPLC buffers such as sodium or potassium phosphate are not compatible.Use the smallest amount of buffer which provides buffering under the analysis conditions (e.g. 10mM). *Select a buffering agent (or modifier) which are within 2 pH units (+/- 1) of the sample's pKa and 2 pH units away from any acid's pKa. 

• For LC/MS applications: Positive ion mode favors acidic mobile phases and Negative ion mode favors basic mobile phases. However, feel free to experiment using both ionization modes and don't forget about using adducts (e.g. ammonium and sodium) with all types of samples to improve signal response. *Maintain these buffers at or below 10 mM. Adjust the pH of the mobile phase to be 1 to 2 units away from your sample's pKa.

Table 1:  Popular examples of useful volatile mobile phase buffers, modifiers and/or additives.

BUFFERING/MODIFIER AGENT                                   USEFUL pH RANGE

• Ammonium formate                                 2.8 - 4.8; 8.2. - 10.2
• Formic Acid                                            3.3 - 4.3
• Pyridine/Formic Acid                               3.3. 4.3, 4.8 - 5.8
• Trimethylamine/Formic Acid                     3.3 - 4.3, 9.3 - 10.3
• Ammonium Acetate                                  3.8 - 5.8; 8.2 - 10.2
• Acetic Acid                                              4.3 - 5.3
• Trimethylamine/Acetic Acid                      4.3 -5.3, 9.3 - 10.3
• Ammonia/Formic Acid                              3.3 - 4.3, 8.8 - 9.8
• Ammonia/Acetic Acid                               4.3 - 5.3, 8.8 - 9.8
• Ammonium Bicarbonate                           5.9 - 6.9,  8.8 - 9.8
• Ammonium Carbonate                              5.9 - 6.9, 8.8 - 9.8  
• Carbonic Acid                                            6 - 8 (pKa 6.37/pKb 7.63)
• 1-Methylpiperidene                                   10.0 - 12.0  

• Trifluoroacetic Acid (TFA)                        pKa = 0.3 (WARNING when used with MS

                                                                                     systems!).  See notes #2 and #4 below.           

*Notes: (1) Formic acid (3.75) is slightly stronger and more volatile than Acetic acid (4.75). Formic acid is often available in higher purity grades and absorbs less in the UV region making it a better choice for most chromatography applications. It works well in positive mode LC/MS analysis, esp at 0.1%. (2) Trifluoroacetic acid (TFA, pKa = 0.3) is very strong and volatile, but we do not recommend its use in LC/MS applications as it can increase the background signal levels (esp. in Negative Mode) LC/MS (m/z 113), be very hard to remove from the source and result in long term instrument contamination. Difluoroacetic acid (DFA) and ammonium formate are other alternatives as they offer good ion pairing capacity with less ion suppression problems. (3) Triethylamine (TEA, pKa 11) is volatile, strong and very stable, but causes similar contamination problems resulting in high background signals when used in Positive Mode LC/MS (m/z 102). (4) Many ion-pairing reagents suppress ionization, bind to the plastics and metals used and contaminate the flow path. If you must use them, please do so using the lowest possible concentrations levels and thoroughly decontaminate the entire flow path of the system after use (or dedicate the MS system to use with them only). Minimize further contamination by labeling and using a dedicated column for the application (Do not use that same column exposed to ion pairing compounds for any other methods or applications). (5) Acids and bases alone provide little "buffering" so should be used with a secondary buffering species to resist change in pH.