Analysis of Mannitol in Human Plasma

Mannitol is an osmotic diuretic that is physiologically inactive in humans and is found naturally in fruits and vegetables as a sugar or sugar alcohol. Mannitol raises the osmolality of blood plasma, allowing more water to move from tissues including the brain and cerebrospinal fluid into interstitial fluid and plasma (1).

analysis of mannitol in human plasma

Fig.1 3D Model of Mannitol

Mannitol can be used to:

  • Reduce Inter-cranial Pressure

Mannitol is administered intravenously and becomes a new solute in the plasma. Thus, increasing the tonicity of the plasma, which will pull water into the intravascular space from the brain parenchyma. The water will then be excreted through the kidneys.

  • Reduce Intra-ocular Pressure

Due to an increase in the plasma's tonicity by mannitol, water from the vitreous humous of the eye is drawn out to the intravascular space.  Finally, the water and mannitol will be excreted by the kidneys. The intraocular pressure is reduced as the vitreous humor’s water content decreases.

  • Promote Diuresis in Acute Renal Failure

Intravenous mannitol is sometimes given to those who have acute renal failure. Even in acute renal failure, the kidneys are responsible for a large portion of mannitol excretion. As mannitol is expelled, it attracts water, boosting the patient’s water output and assisting in the prevention or treatment of fluid overload caused by oliguria in acute renal failure.

  • Excrete Toxic Materials

Mannitol can be used to promote the excretion of harmful chemicals, substances, and medications, similar to how it is used to treat oliguria in acute renal failure.

  • Prevent Intradyalytic Hypotension

Hemodialysis patients frequently experience intradialytic hypotension and dialysis disequilibrium symptoms. The reason for this is a decrease in intradialytic osmolality. By increasing serum osmolality, mannitol can help prevent intradialytic hypotension.

Administration

Mannitol is administered intravenously when used for medical purposes (2). Mannitol
is available in a variety of quantities, ranging from 5% mannitol (5 gm mannitol
dissolved in 100 mL solution) to 25% mannitol (25 gm of mannitol dissolved in 100 mL
of fluid) (2). The amount of mannitol administered depends on the type of condition
that is being treated (2).

  • Increased Intercranial Pressure – dosages range from 0.25 g/kg to 2 g/kg
  • Increased Intraocular Pressure – dosages range from 0.25 g/kg to 2g/kg
  • Oliguria treatment – 0.2 g/kg
  • Excretion of toxic materials – 0.25 g/kg to 2 g/kg

Side effects

  • Heart failure
  • The movement of free water into the intravascular region might cause or worsen electrolyte imbalances. Hyponatremia, hypokalemia, and hypocalcemia are some of the electrolyte disorders that might occur.
  • Worsen cerebral edema. Frequent mannitol doses can cause mannitol to penetrate the blood-brain barrier, worsening cerebral edema because the mannitol that has leeched into the brain attracts water into the brain rather than out of it.
  • Dehydration due to diuresis

Method

We have analyzed mannitol in human plasma. Using our HPLC instruments (Fig. 2), we have determined our LLOQ and ULOQ to be 7.5 and 1500 µg/ml, respectively.

HPLC mannitol analysis

Fig. 2 HPLC

To book our instruments please contact us here.

References

1. National Library of Medicine. Mannitol. (Internet) (Cited on Aug 2021) Available from: https://pubchem.ncbi.nlm.nih.gov/compound/Mannitol

2. StatPearls. Mannitol. (Internet) (Cited on Aug 2021) Available from: https://www.ncbi.nlm.nih.gov/books/NBK470392/

Our HPLC Training Courses

Looking for A HPLC training COURSE?

High Performance Liquid Chromatography is a powerful analytical tool. We offer HPLC training courses which will give you all the skills needed to master the HPLC instrument.

Who is this course for?

HPLC training

Fig.1 Our HPLC system

HPLC training is for any person (no previous experience needed) who is looking to improve their laboratory skills or learn how to master the HPLC instrument. Depending on your previous experience we will prepare a tailored training to cover everything you may need to know.

Both HPLC training courses are taught on a 1 to 1 basis (maximum 2 people) so that you can take the most out of this training and have plenty of time to adapt to the instrument. We focus on giving you a complete and practical experience, hence by the end of the training, you will feel confident to run by yourself your own set of samples on the instrument.

Course Options

We offer two HPLC courses:

  • 3 Day HPLC Training
  • 1 Day HPLC Refresher Course

The 3 day course covers all the HPLC basics, standard laboratory training, sample preparation, method development and data analysis. It is the perfect choice for someone that may want to learn how to set and run HPLC to improve their analytical skills. This is a complete course that aims to teach you all the fundamentals you need to run your own samples.

The 1 day course covers HPLC basics and method development, however it is subject to the trainee’s previous experience. This course is advised to someone that already has some experience with HPLC and wishes to remind themselves about HPLC basics/method development or is looking for advice on method development for their own research project.

Structure of the 3 Day Course

  • Day 1 – Essential reading & standard laboratory training
  • Day 2 – Sample preparations & method development
  • Dat 3 – Method development (continuation) & data analysis

At the end of the course, there is a Q&A with our manager, you can clarify any questions you might have.

How to book

Fill out the form in the contact us page, or if you prefer you can email us or call us directly. Please click here to book your HPLC training course!

 

 

 

 

Amlodipine in Veterinary Practice

Amlodipine has been used to treat high blood pressure in humans, but it has now been approved for veterinary practice. It is given to cats and dogs to treat hypertension and other associated diseases.

About Amlodipine

This drug is classified as a calcium channel blocker. The heart and arteries contract more forcefully when calcium is present, thus calcium channel blockers relax and widen blood arteries by inhibiting calcium. This effect will lower the blood pressure which prevents heart diseases, heart attacks and strokes.

amlodipine in veterinary practice

Fig.1 Chemical Structure of Amlodipine

Side effects

Like all medicines, amlodipine can have side effects. In animals, common side effects include vomiting, lack of appetite, diarrhea and sleepiness (1). Serious side effects such as severe weakness, changes in urination, abnormal bleeding or rapid heart rate are rare, but can also occur (1).

Hypertension in Cats

Hypertension in cats is a moderately frequent and possibly fatal condition. The most prevalent kind of hypertension in cats is secondary hypertension, which is caused by an underlying systemic condition that impairs the body’s regulatory mechanisms responsible for maintaining normal blood pressure (2). Among these conditions, chronic kidney disease and hyperthyroidism are the most common (2).

Injury to organs can happen due to hypertension. The most prevalent target organ injury found in cats with hypertension is ocular disorders, which can range from retinal detachment resulting in blindness to more subtle indications like hemorrhage or swelling in the back of the eye (2).

Hence, not only can amlodipine treat hypertension, but it can also be given to cats to prevent damage to kidneys, heart, brain, and eyes.

Analysis of Amlodipine in Feline Plasma

Using our HPLC system and LC-MS, we have examined amlodipine in feline plasma. The LLOQ and ULOQ were set for the method as 0.5 ng/ml and 100ng/ml respectively for Amlodipine. The Limit of Detection was calculated as -0.001 ng/ml in feline plasma.

References

  1. Rania Gollakner. Amlodipine. (Internet) VCA. (cited on 2021 Jul) Available from: https://vcahospitals.com/know-your-pet/amlodipine-besylate
  2. Cornell Feline Health Center. Hypertension: Feline hypertension. (Internet) College of Veterinary Medicine. (updated 2021; cited on 2021 Jul) Available from: https://www.vet.cornell.edu/departments-centers-and-institutes/cornell-feline-health-center/health-information/feline-health-topics/hypertension

Developing Good Pipetting Techniques

hOW IMPORTANT IS IT TO DEVELOP GOOD Pipetting Techniques?

In order to achieve optimal and reliable results for our diagnostic services we ensure all staff are
trained to a high degree in basic laboratory skills, such as liquid handling.

Pipettes are used daily in our lab for liquid handling. Two factors contribute to good laboratory
pipetting: tools and techniques. At the Bio-analysis centre we use mainly single channel manual
‘transferpette’ pipettes (Figure 1) which are known to be highly accurate at various volumes; however, the
most important factor is the expertise of the operator.

developing good pipetting techniques

Fig. 1 Single Manual Channel Transferpette

There are two main pipetting techniques: forward pipetting and reverse pipetting (/back pipetting).
Forward pipetting aspirates from the first stop and ejects until the second, whereas reverse
pipetting aspirates from the second stop and ejects until the first.

Other factors include:

– Immersion angle and depth
– Pre-rinsing
– Speed
– Temperature of environment and samples
– Performing pipette leak testing daily

Prolonged poor ergonomics while using pipettes can result in repetitive strain injuries (RSI) and
carpel tunnel syndrome (CTS) (El-Helaly et al., 2017). Studies have shown how higher work duration
increases the risk of developing RSI and CTS (El-Helaly et al., 2017, Bjorksten et al., 1994) . During our
training courses we recognise this issue and work with you to improve your pipetting technique
giving you the confidence to work comfortably in a laboratory environment.

Good laboratory pipetting is fundamental in our work producing accurate and precise data from the
HPLC and MS-LC. We are experienced in pipetting difficult solutions such as plasma and serum,
which are notoriously bubbly and therefore difficult to pipette!

Click here to find out more about our 1 day lab skills course.

References:
BJORKSTEN, M. G., ALMBY, B. & JANSSON, E. S. 1994. Hand and shoulder ailments among laboratory
technicians using modern plunger-operated pipettes. Appl Ergon, 25, 88-94.

EL-HELALY, M., BALKHY, H. H. & VALLENIUS, L. 2017. Carpal tunnel syndrome among laboratory
technicians in relation to personal and ergonomic factors at work. J Occup Health, 59, 513-
520.

Liquid Chromatography Method Development

Why is Method Development important? 

In the pharmaceutical sector, liquid chromatography procedures are used to assay compounds and quantify impurities present within medicinal products. Method development allows the creation of the best method to test for impurities and compounds which help ensure the quality, safety and efficacy of new products and drugs.

Liquid Chromatography Method Development 

Method development is the process by which the optimal conditions are found for a particular compound to separate it from any contaminants. To design the best method for a particular sample, the following parameters and conditions must be optimized:

  • Selection of chromatographic mode (Reverse Phase, Normal Phase,
    Size exclusion, HILIC, etc.)
  • Selection of stationary phase (C18, C8, Phenyl-hexyl, Biphenyl,etc.)
  • Selection of detector (UV, RID, MS, ELSD)
  • Selection of mobile phase (which buffers; pH of buffers)
  • Flow rate
  • Preparation of samples (protein precipitation, Solid Phase Extraction,
    etc)
  • Injection volume
  • Concentration
  • Calibration Range

HPLC Method Development

Our Nexera XR HPLC system (Fig.1) is ideal for developing procedures for a variety of material, ranging from complicated combinations of tiny molecules or proteins to single chemical purification.

HPLC method development

Fig. 2 Nexera XR HPLC System

Mass Spectrometry Method Development

We have the expertise to design methodologies to quantify most drug-like substances using our Shimadzu LCMS-8040 (Fig.2).

Mass spectrometry method development

Fig.2 Shimadzu LCMS-8040

Our manager Dr. Carolyn Hyde has over 20 years of experience developing methods on HPLC and LC-MS. Click here to contact us for more information on our services. 

References

Avoomeen. Why Pharma Companies Must Invest in Method Development & Validation (Internet). (Cited on 16th July) Available from: https://www.avomeen.com/pharma-companies-invest-method-development/

 

 

 

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.