ANALYSIS OF PREGABALIN

What is Pregabalin?

“Pregabalin is used to treat epilepsy and anxiety, it is also taken to treat nerve pain that can be caused by different illnesses including diabetes, shingles, or injury.

Pregabalin works in different ways:

1) in epilepsy it stops seizures by reducing the abnormal electrical activity in the brain

2) with nerve pain it blocks pain by interfering with pain messages travelling through the nervous system, through the brain and down the spine

3) in anxiety it stops your brain from releasing the chemicals that make you feel anxious

Like most medicines it comes with side effects even when following your prescribed amount, in rare cases it can lead to hallucinations, difficulty breathing and thoughts of self-harming”(1)

Our Method

We were asked to look for Pregabalin in canine plasma as part of research into treatment of dogs with epilepsy.

The Lower Limit of Quantitation (LLOQ) and Upper Limit of Quantitation (ULOQ) were established for the method as 1ng/ml and 1000ng/ml respectively, and the Limit of Detection was calculated as 0.014891ng/ml in canine plasma.

The system linearity was determined by the evaluation of Pregabalin standard curves analysed on 3 separate days. The curves were fitted using linear regression with 1/C2 weighting. Each standard curve consisted of 11 standards ranging from 0ng/ml to 1000ng/ml.

We were able to measure levels of pregabalin over the treatment period and hence show the level rise then fall over the 12 hour period.

References

(1) Pregabalin: medicine to treat epilepsy and anxiety [Internet]. nhs.uk. 2020 [cited 22 December 2020]. Available from: https://www.nhs.uk/medicines/pregabalin/

PRAVASTATIN ASSAY

The Analysis of Pravastatin in Human Plasma

Chemical structure of Pravastatin. Source: https://en.wikipedia.org/wiki/Pravastatin

What is Pravastatin?

“Pravastatin belongs to a group of medicines called statins, it is used to lower cholesterol if you’ve been diagnosed with high blood cholesterol, it is also taken to prevent heart disease including heart attacks and strokes” (1)

1 in 1000 people taking pravastatin may have serious side effects such as: muscle pain, tenderness, weakness or cramps, severe stomach pain. In rare cases its possible to have a serious allergic reaction (anaphylaxis).

Method:

The first objective of this project was to validate a method for the quantitation of pravastatin residues in human plasma by LCMS analysis.  The second objective was to validate a method for the quantitation of pravastatin lactone in human plasma by LCMS analysis.

The LLOQ and ULOQ were established for the method as 1ng/ml and 100ng/ml respectively for Pravastatin and defined as the lowest and highest validated concentration that meet all acceptance criteria. Accuracy and precision were established at the LLOQ.
.The Limit of Detection for Pravastatin was calculated as 0.885654ng/ml in human plasma.

It was not possible to validate the method for Pravastatin Lactone as it was too unstable, however the Limit of Detection was established to be 11.81485ng/ml in human plasma.

This project took seven years from initial discussion to receipt of samples, so patience is sometimes required!

References.

(1) Pravastatin: a medicine used to lower cholesterol [Internet]. nhs.uk. 2020 [cited 13 November 2020]. Available from: https://www.nhs.uk/medicines/pravastatin/

WHAT ARE CANNABINOIDS?


They are chemicals found in cannabis, the most notable is phytocannabinoid tetrahydrocannabinol (THC), the primary psychoactive compound in cannabis plants. Cannabidiol (CBD) is another major constituent of the plant and these are just two of at least 144 known different cannabinoids isolated from cannabis all exhibiting various affects.(1)

Why use CBD?

CBD oil has been studied for its potential medical role in easing symptoms of many common health issues including anxiety, depression, acne, and heart disease as well as being put into various non-medical products from beers to soaps.(2)

How can B-AC help?

Say a product such as soap has a claimed level CBD, we have an assay to determine the actual level of CBD present, as well as checking that the level of THC is below the legal limit.

What are the legal restrictions?

There is no maximum level of CBD so long as it cannot be easily separated from other compounds in the product, however your product can only contain 0.01% (not detected) THC and no more than 1mg in any CBD product.(3)

The packaging must have clear labelling; no medical claims; its CBD content; manufacturing details and ingredients. This is vigorously tested to ensure companies are not lying about the THC content as it can have the most dangerous psychoactive affects.

Examples

A 200g block of soap has been manufactured with 100mg of CBD and 20mg of THC, are these levels acceptable for it to be sold?

CBD

200g:100mg

200,000mg:100m

(100/200,000) x 100 = 0.05% or 500ppm

THC

200g:20mg

200,000mg:20mg

(20/200,000) x 100 = 0.01% or 100ppm

 

That level of THC is above the legal requirement as there are traces of other cannabinoids so can be no higher than 1mg:

(0.5/200,000) x 100 = 0.00025 or 2.5ppm

The THC levels would need to be tested at an accredited lab to ensure they are at the legal and displayed level.

 

What can we do for you?

We have an assay which can determine the level of the 13 main cannabinoids. We can also develop methods for synthetic or unusual cannabinoids unique to your requirements.

 

HPLC graph with 13 peaks for tested cannabinoids

 

Regulations

https://www.theextract.co.uk/is-cbd-legal-uk/

References:

1) Cannabinoid [Internet]. En.wikipedia.org. 2020 [cited 11 September 2020]. Available from: https://en.wikipedia.org/wiki/Cannabinoid

2)  7 Benefits and Uses of CBD Oil (Plus Side Effects) [Internet]. Healthline. 2020 [cited 11 September 2020]. Available from: https://www.healthline.com/nutrition/cbd-oil-benefits#:~:text=CBD%20oil%20has%20been%20studied,for%20pain%20and%20symptom%20relief.

3) CBD Regulation UK – – Explained – – Medic Pro Limited [Internet]. Medic Pro. 2020 [cited 11 September 2020]. Available from: https://www.medicpro.london/cbd-regulation-laws-uk/#:~:text=So%20the%200.2%25%20limit%20does,limit%20of%20detection%20of%200.01%25.

 

GUT MALABSORPTION ASSAY

Gut permeability is an issue when there are changes and the absorption of nutrients is altered.

The Theory:

A solution of 3 sugars are given to patients at a set concentration, then either urine or blood samples are taken at set time points for analysis. The ratio of the Lactulose (disaccharide) to either Rhamnose or Mannitol (monosaccharides) indicates if and where there is a problem in the gut. These 3 sugars are not metabolised by the body, they just permeate across the gut wall.

How we are involved:

We have developed two analysis methods, based on published work, to measure these sugars in either urine or plasma. We have a lower limit of quantitation of 15µg/ml in Urine and 7.5µg/ml in Plasma, however we would advise that the actual lower levels should be 25µg/ml for urine and 15µg/ml for plasma to ensure that the lowest levels do not fall outside the calibration range. Figure 1 shows the timing and peaks for the sugars being sent round the HPLC.

Figure 1: Graph from the HPLC showing the 3 sugars separation and their associated retention times.

We are working with Mike Hewetson from the Royal Veterinary College to adapt the plasma method for use with equine serum. At present, there is no easy way of determining where a problem is in the equine gut and it is hoped that this test will offer an alternative to submitting the horse for a surgical procedure.