How to make sure your results are reliable

By Keith Williams, Ph.D. - Business Manager, Analytical Standards Europe, Cayman Chemical

So you have purchased or are about to purchase a reference material or analytical standard from Cayman. Before you do, it may be useful just to stop and think for a few moments about how you are going to use it. Our products are carefully manufactured to deliver the best possible product to analytical laboratories. However, no matter how carefully we synthesize and characterize them, if used incorrectly, they may deliver poor analytical data and ultimately, poor results. What are some things you should think about before purchasing and ultimately opening the reference material or analytical standard for the first time?

Purchasing the Correct Reference Material

Why are you purchasing the standard in the first place? It is likely that you have come across the analyte in your work, and you are looking to either confirm the identity of your target or determine the amount you have present. Normally, some form of method validation/verification/re-verification processes is also involved.

Most organisations have a quality management system that clearly defines what you need to do to confirm that your reference material meets your testing requirements. There are also international standards and guidelines that can help in this evaluation. Many international guidelines for quality, including ISO/IEC 17025 and international forensic guidelines, state you can use reference materials produced under ISO 17034 without further evaluation for either calibration or control purposes. Our Certified Reference Materials (CRMs) and Reference Materials (RMs) characterized and produced under both ISO/IEC 17025 and ISO 17034 fully meet these stated requirements and, in almost every case, can be used without further verification.

The responsibility for the correct identification of an appropriate reference material will always lie with the laboratory user. Only the laboratory user can know if the specific reference material selected is fully fit for their analytical purposes.

Receipt and Storage of Unopened Materials

When you receive the standard, you should check that all the bottles and ampoules are unbroken and ensure that you have no concerns regarding the integrity of the delivery package. Things to look out for include damaged packaging, missing documentation, and any indication that the products may have been exposed to higher temperatures than expected. If you think that there may be an issue, contact Cayman Chemical as soon as possible. Where practical, include photographs. We can then advise the best way forward with the products in your possession.

Expiry Date

As simple as it may seem, you should always check the expiry date on the product prior to opening the vial or ampule. Manufacturers generally will guarantee the quality of reference products until the day the vial or ampoule is opened for the first time as long as:

1. The product is stored under the recommended conditions.
2. The product is opened before the listed expiry date.

We include the expiry date on each Confirmation of Analysis or Certificate of Analysis (CofA). In addition, we continually monitor the products at our production sites in case there are any subtle but significant changes.

It is also good practice to check our website to see if an updated CofA has been released for the batch you are using. This may extend or, on very rare occasions, reduce the expiry date of a particular product and/or a specific batch. Once the vial or ampoule is open, all responsibility for stability moves from the producer to the individual laboratory.

It is good practice that any dilution created from the original product is labelled with at least the date, an identifier of who created it, the concentration, and the expiry date. In many cases, internal quality systems will demand this as well as additional information to be fully compliant.

Any unused solid product should be stored as per the original manufacturer’s instructions. Any open solution should be placed in a tightly closed vial with the original date of opening noted. Any dilution or working standard should be kept under defined storage conditions that should reflect those required for the original product. Please note that CRMs are one-time use, and therefore, we don’t provide long-term storage recommendation once the ampules are snapped. RMs, when stored at recommended storage temperatures, should undergo periodic testing to ensure stability.

A Note on Overfilled Vials

Most reference material manufactures are generous. They will provide you with more product in the vial to make sure that you can recover the 1 ml, 10 mg, or 100 μl that you ordered. In some cases, there can be 10% or more extra material in the purchased container. Therefore, it is best practice to transfer a known amount of the material from the supplied vial or ampoule to a secondary container and dilute down your working solutions from there. This can be done in many ways but the two most common are:

1. Aliquoting: This is where a known portion of the material is taken from the supplied reference material and transferred to a second container. This can be done using a balance (for solid materials) or a pipette (for solutions). Care must be taken in choosing the correct balance or pipette to give the end user the required accuracy and precision.
2. Total recovery: For solid materials, the contents are recovered from the original bottle by dissolving in an appropriate solvent and quantitatively transferring the solvent to a second container. This will allow maximum recovery of the reference material but also uses up all the reference material in a single operation. Because the shelf life of the solid reference material you have purchased was assessed in its original form, it is not safe to extrapolate that shelf life to a solution you have created in your laboratory. The total recovery method, if executed correctly, is a robust method for creating a solution of known concentration. Just note that since the exact amount in the vial is unknown, the known concentration is strictly nominal unless a pre and post tare of the vial is done as part of the transfer. If you chose to use the total recovery method, you should consider the cost implication of consuming the whole product in the preparation of a single standard.

Considerations for Purity, Salt Forms, and Water of Crystallisation

In certain instances, such as for solid products, you may need to take account of the salt form, yet for our CRM solutions this should not be necessary. The concentration of all our CRMs in solution are stated in the free base form. There is no need to correct for the presence of salt, purity, water of crystallisation, or similar.

Our solid products, however, need to be checked on a case-by-case basis to consider salt forms and other factors. You should divide the weight of the freebase form of the substance by the combined weight of the substance and the salt. This will give you a correction factor (always between 1 and zero). Multiply the dispensed weight by the correction factor and that will give you the concentration of free base drug you have.

This critical step, if done incorrectly, can result in an overestimation of the true concentration of the substance you are measuring in a specific sample.

For example, amphetamine (hydrochloride) has a free base molecular weight of 135.20 and a combined substance plus salt weight of 171.67. Using a dispensed weight of 10.6 mg, the free base correction factor would be 135.20 ÷ 171.67 = 0.788. The free base concentration could then be calculated as 10.6 mg x 0.788 = 8.35 mg.

Handling Sticky Materials

If only all reference materials were nice white powders or crystals. While many are, there are a significant number that are not. Several key cannabinoids, for example, are sticky oils in their pure forms. These materials can be dispensed by using either of the two methods described above. But when handling small quantities, perhaps it is best to use the total recovery method. It is not unheard of for laboratory staff to open a vial containing 10 mg of an oily reference material and think that there is nothing there, not realising that the sticky liquid on the vial cap is what they are looking for. Using a carefully chosen solvent will maximise the recovery of the substance.

Alternatively, we know that many of these sticky substances can be problematic, so we also make them into solutions ready for you to use.

Choosing a Solvent

The impact of your choice of solvent to dilute down your standard is sometimes underestimated. Although it sounds obvious, make sure that your solid reference material is soluble in your chosen solvent. We all probably know more than one individual who has made that mistake. Secondly, make sure that your drug is stable in the solvent. The breakdown of drugs in solution is not always fully considered. Finally, if making a mixture of several different reference materials, don’t forget to check that the solvents you chose are miscible, don’t interact in some form, and that all the analytes are stable when placed together in solution.

International Standards and Recommendations for Preparing In-House Quality Control Materials

There are extensive guidelines, recommendations, and agreed-upon international standards on how laboratories should carry out analysis, and there are many published papers on what organisations should do to validate methods. When it comes to how a laboratory should prepare calibration or control materials, there are surprisingly few clear and concise guidelines. This can at least be partially attributed to individual laboratories having differing requirements and quality systems. This lack of true interlaboratory consistency has potential to impact the reporting of the true analytical value of a sample when analysed by different laboratories.

One of the few documents available to offer guidelines is ISO Guide 80. Released in 2014, it clearly and informatively outlines what laboratories should do when preparing what the authors define as in-house Quality Control Materials (QCMs). The concepts and advice provided probably reflects what many organisations already do, but formalises it in a clear and concise manner, illustrated by a number of nicely worked examples at the rear of the document.

Summary

In any analysis, the Cayman reference materials and analytical standards being used to directly measure the target analyte, though likely one of the lowest cost items in the whole analytical process, provide the greatest value to the end user. They are produced by a team who take great pride in their manufacture and qualification, with more than 500 years of accumulated experience in metrological, analytical, and synthetic chemistry. When used correctly, Cayman Chemical’s reference materials and analytical standards give you confidence that you are producing valid analytical data and issuing correct reported values to your customers.

Further Reading

ISO/IEC 17025:2017 - General requirements for the competence of testing and calibration laboratories

ISO 15189:2012 - Medical laboratories -- Requirements for quality and competence

ISO Guide 80:2014 - Guidance for the in-house preparation of quality control materials (QCMs)

Guidelines on the Use of Reference Materials in Forensic Drug Analysis, ENFSI (PDF)

SWGDRUG Recommendations Edition 7.1 (2016-June-09)