Technical Guide

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What is ISO Guide 34?

ISO Guide 34:2000 outlines the general requirements for the competence of reference material producers. This guide is used as the basis for accreditation of the producers. A copy of the ISO Guide can be purchased from your national standardisation body.

Do all reference materials need to be sourced from an accredited producer?

Laboratories must have confidence in the competence of the producers from whom they obtain their reference materials. The basis of this confidence should be demonstrable to regulatory authorities and ISO Guide 34 accreditation is considered appropriate for this.

Do I need two sources of ISO Guide 34 material to satisfy accreditation requirements?

One source of ISO Guide 34 reference material is sufficient to satisfy accreditation requirements. However, use of two independent sources produced in accordance with ISO Guide 34 provides an additional level of confidence, to both user and assessor, that the two will agree. The cost of consumables, analyst and instrument time required to repeat failed assays significantly exceeds the cost of using two independent ISO Guide 34 reference materials.

Do calibrators and quality controls need to come from separate sources of reference material?

Several ISO guidelines discuss the requirements for independent sources of reference materials for calibration and control:

  • In section 5.13, note 6 of ISO/IEC Guide 99:2007 (VIM) it states that 'in a given measurement, a given reference material can only be used for either calibration or quality assurance'.
  • In note 4 following the definition of reference material that ISO REMCO adopted in 2005 it states, 'A reference material can only be used for a single purpose in a given measurement'.
  • ISO Guide 31- In the January 2012 this note has been modified to read, 'A single reference material cannot be used for both calibration and validation in the same measurement procedure'.

In some instances it is difficult (or even impossible) to find two independent sources of reference materials. UKAS acknowledges this practical problem. They advise:

  • If the same source/batch of material is used for both calibration and quality control, measures must be in place to ensure that the possibility of contamination is not only considered, but that measures are in place to detect this. Any bias in the reference material used for calibration or quality control must be detectable by the quality control measures in place in the laboratory.'

As an ISO:17025 and ISO Guide 34 accredited producer and distributor of key reference materials suppliers throughout the world, Clearsynth standards are in a perfect position to offer independent sources of reference material for calibration and control.

My reference material was accompanied by a certificate, does this mean it's a certified reference material?

Most manufacturers provide some form of documentation with their products. In some instances this may be referred to as a 'certificate'; however this does not necessarily mean that it fulfils ISO's requirements for a certified reference material. The certificate must be issued by a technically competent body, i.e. National Metrology Institute (NMI) or an ISO Guide 34 accredited producer. ISO Guide 31 and ISO 15194:2002 both specify requirements for the description of reference materials and the contents of certificates and labels.

The information deemed essential on a certified reference material certificate, includes:

  • Name and address of the manufacturer
  • Material identification, including batch/lot number
  • Description of the material
  • Intended purpose
  • Instructions for use
  • Storage conditions
  • Product certification, including full description of methods used and uncertainty
  • Date of document issue and material expiry date

What is the difference between a certified reference material and a chemical?

According to the ISO/IEC Guide 99:2007, International Vocabulary of Metrology (VIM), a certified reference material is a material of known homogeneity and stability, which is accompanied by documentation (certificate of analysis), issued by an authoritative body, providing one or more specified property values. These values have associated uncertainties and trace abilities obtained using valid reference measurement procedures. A certified reference material has been established to be fit for its intended use in measurement. In the laboratory a certified reference material is typically classified as a pure substance used to prepare calibration solutions or matrix material used to evaluate measurement bias.

Chemicals are occasionally mistaken for reference materials. These are typically reagent grade or synthetic precursors. Documentation listing the basic physical properties may accompany the material. This documentation may even be referred to as a certificate; however the materials do not comply with ISO standards requirements for certification and should not be assumed to be certified reference material. The purity of chemicals is usually given as a minimum concentration i.e. =98%. These should not be used for quantitative purposes 'as is'. They may be suitable for use as secondary reference materials providing the purity is verified by comparison to a certified reference material.

Ref: ISO/IEC Guide 99:2007 International vocabulary of metrology - Basic and general concepts and associated terms (VIM):

Ref: Eurachem Terminology in Analytical Measurement Introduction to VIM 3. First Edition 2011:

Ref: European Network of Forensic Science Institutes Drugs Working Group (ENFSI) Guidelines on the use of Reference Materials in Forensic Drug Analysis

I can't find a reference material of the drug I need.

We have years of experience in reference materials, combined with a network of European offices and relationships with key reference materials suppliers throughout the world. We may already have a source of the material you are after. If you are looking for something that doesn't appear on the web shop or in the catalogue, email us at If we are unable to locate a commercial source of the material you require, we will offer a custom synthesis service.

Whether undertaken by us or any other supplier, custom synthesis can sometimes be a costly route. Commercial suppliers need to be convinced that production costs can be recovered through sufficient sales, a difficult task given the unpredictable and rapidly developing market. We routinely document and collate customer requests from around the world. Multiple requests are important in helping us assess demand for reference materials. If you are interested in a certain reference material the chances are someone else will be too!

Can I use a legal high purchased from a head shop?

Purchasing 'reference materials' directly from legal high outlets can seem an appealing choice, given commercial delays in the provision of reference materials and associated costs. In fact so called 'grey-market sourcing' of materials and conversion into reference materials is one route used by commercial suppliers in the production of standards. However, this approach is fraught with problems.

The legal high market is unregulated and published studies have shown that products do not necessarily contain what they report to contain. Content has also been shown to differ on repeat purchase of the same material. Manufacturers often claim high percentage purity, and initial examination via GC-MS may appear to concur. However, further examination by techniques such as NMR and CNO, which are not widely available in most laboratories, can tell a very different story. At best, after purification you may end up with a fraction of the product you purchased. At worst you may incorrectly identify or quantify your unknown.

Furthermore products sold as 'legal highs' have even been found to contain one or more controlled substances. Depending on the legislation within your country you may be liable to prosecution if found in possession of these products, irrespective of whether or not you have knowingly purchased a controlled drug.

Why are reference materials available as both powders and solutions?

The CLEARSYNTH range produced byus contains both powder and solution reference materials. For most applications either is suitable, however there are advantages and disadvantages associated with the different presentations:

Many people prefer solutions as this removes the laborious process of having to weigh the material themselves. It also eliminates any in-house errors associated with correction for purity, salt or water content. Plus the solubility of the material has already been investigated, so you can be confident that it has been dissolved in an appropriate solvent. In accordance with ISO/IEC 17025 and ISO Guide 34, the uncertainty of the concentration is reported for all of our solutions, and incorporates contributions from the purity, material density, balance and weighing technique.

We acknowledge that some reference materials can be expensive. Vials containing low milligram quantities, i.e. 1 milligram may make a material more affordable, but are impractical in terms of weighing for quantitative purposes. Accurately calibrated, lower concentration solutions are one way in which we can make available to you, the high quality materials you want at prices you can afford.

Despite the advantages of solutions over powders, some people still prefer powders. Powders may be more applicable for certain applications, such as fourier transform infrared spectroscopy (FTIR). Powders are also more appropriate for drugs that are unstable in solution.

Why are some reference materials sold as free base and others as salts?

Our range of reference materials contains products supplied in various forms. Salts (hydrochloride, sodium, sulphate etc) and hydrates (addition of water) help to stabilise or increase the solubility of the parent compound. The term hydrate is used to describe a one to one ratio of water to drug. Dihydrates and trihydrates are used to describe two to one and three to one ratios respectively. Errors can occur in quantitative analysis through failure to correct for salts and hydrates. The concentration of all our solutions are corrected to the free base (or free acid) concentration. Corrections are required for some, but not all powders. This is achieved by converting the actual weight of the material by the percent of drug present in the weighted material.

Our powders and solutions are overfilled to ensure a minimum of 10 milligrams or 1 millilitre respectively.

Powder: It can sometimes be difficult to recover all the powder from a vial. The wide mouthed vials are designed to assist in gaining access to the material; however it can distribute across the surface area of the vial or lid. Before opening, tap the vial gently on the work surface so that any powder distributed on the lid or sides of the vial during transit collects at the bottom. Weigh the material using a 5 place analytical balance. Depending on the nature of the material being weighed, use of specialist anti-static functions on the balance can assist. Total transfer (dilution of the reference material in situ) is advisable when dealing with very small quantities of reference material. Weighing the container before and after material dilution and transfer will enable calculation of the total weight of standard removed from the vial. If necessary an exact calibrated weight of powder per vial can be obtained for our product range.

Solutions: Before opening ampoules hold them by the top and flick them gently to ensure that all of the liquid is at the bottom. Due to over-filling laboratories are advised to use measured volumes of the standard solution before diluting to the desired concentration.

What is best practice for Labelling reference materials and derived solutions?

To assist reference material producers and promote harmonisation of practice, ISO Guide 31 specifies requirements for the contents of reference material certificates and labels. These requirements also feature in section 5.7.5 of ISO Guide 34, which in addition addresses the physical aspects.

"The reference material label shall be securely attached to the product container of an individual reference material units, and shall be designed to remain legible and intact under defined storage and handling conditions within the lifetime of the material".

The labels relate only to reference material units to which they are attached. The label should not be transferred from the reference material to any secondary container or derived stock or sub-stock. Additionally labels should not be placed on top of, or obscure any information from, existing labels.

It is recommended that labels on in-house prepared stocks or sub-stocks should include, but not necessarily be restricted to, the following information:

  • A unique reference name/number or description of contents
  • Concentration of contents
  • Storage requirements
  • Date of expiry or re-test
  • Ideally, labels should be waterproof and suitable for fridge/freezer storage, and the use of indelible ink will help to ensure the integrity of the information documented.

What is CLP and GHS?

On the 20th January 2009 new regulations ((EC) No. 1272/2008) for Classification, Labelling and Packaging (CLP) of substances and mixtures came into force to harmonise hazard communication. These were based on the United Nations' Globally Harmonized System of classification and labelling of chemicals (GHS) regulation. Irrespective of quantity, producers who introduce a hazardous substance on the market need to label and package in accordance to CLP. Furthermore, ISO Guide 34 specifies that labels shall also "comply with requirements related to safety and risk regulations." The exact requirements for label contents is dictated by the physical size of the reference material, but at a minimum the label should contain hazard pictograms, the product identifier and name and telephone number of the supplier. All CLEARSYNTH products fully satisfy National and International labelling for transportation and health and safety.

Can CLEARSYNTH reference materials be re-used after the seal has been broken?

Unless otherwise stated our powder reference materials are not air or moisture sensitive. In such cases, opening the vial will not alter the product and the associated assay value. However, we can only guarantee integrity of our reference materials until first opening of the vial or ampoule. It is the responsibility of the user to ensure integrity afterwards by correcting further storage and handling of the reference material.

This is set out in section 5.7.4. of ISO Guide 34, which specifies that;

'The reference material producer shall ensure that the integrity of each individual reference material unit is maintained until the seal has been broken or up to the point when presented for analysis. The producer cannot be held responsible for the material once the seal has been broken.'

I'm developing a new method and am unsure of what internal standard to choose?

Internal standards are used to compensate for loss of analyte during sample preparation, and variation in mass spectrometric analysis (i.e. ionization efficiency). The assumption is that internal standard losses will be similar to losses of analyte. An ideal internal standard should fulfil the following criteria.

  • Not already be present in the sample
  • Have similar chemical and physical properties to the analyte
  • Extract and derivatives in the same way as the analyte
  • Elute close to the peak of interest
  • Be free from interferences
  • Have a similar response to analyte
  • Be stable

Removal of CLEARSYNTH ampoules from packaging

It is difficult to find a happy medium between ensuring ampoules are packaged securely for transit, and enabling them to be easily removed from the packaging on receipt. Likewise this balance exists between ensuring the ampoules themselves are strong enough not to break unintentionally, yet easy and safe to open at the predetermined breaking point when required. To minimize risk of breaking open the ampoules prematurely whilst removing them from the packaging, we recommend sliding them out rather than using the top of the ampoule as an anchor point for removal. The latter approach puts stress on the neck of the ampoule thereby increasing risk of premature breakage. Alternatively pressing gently on the outside of the packaging at the body of the ampoule to dispense it in a tablet fashion, or using the label to pull it out, if exposed, also helps to prevent breakage.

Long term storage conditions

We recognize that fridge and freezer space in a laboratory is often at a premium and that there are logistical challenges associated with the storage location of reference materials. For this reason our certificates detail the minimum specified storage requirements and those under which real time stability testing is performed. Unless otherwise stated, storing reference materials in organic solvent at temperatures below those specified is not detrimental, and in many instances can improve long term stability. Products in aqueous solvent should not be stored at temperatures below 0°C unless explicitly stated.

How stable are my reference materials after opening?

Unfortunately we are unable to advise on expiry and retest dates of reference materials, or subsequent working solutions, after opening. It is the responsibility of the user to ensure the integrity of the material once the seal has been broken, as there are too many laboratory specific variables (usage pattern, container, storage conditions, volume, dilution, solvent/matrix etc).

This view point is reinforced by section 5.7.4. of ISO Guide 34 (General requirements for the competence of reference material producers), which specifies;

'The reference material producer shall ensure that the integrity of each individual reference material unit is maintained until the seal has been broken or up to the point when presented for analysis. The producer cannot be held responsible for the material once the seal has been broken.'

Ideally solutions should be used shortly after opening the ampoule to avoid concentration changes due to evaporation. Likewise, powders should not be stored for any length of time after opening, due to the risk of water absorption from atmospheric humidity.

There are a number of methods used by laboratories for assessing the integrity of a reference material after opening. The frequency or approach may alter depending on the drug and its properties.