The Scottish Health Survey 2024 - Volume 2: Technical Report

Chapter 3 - Quality Control of Saliva Analytes

Victoria Wilson, Scottish Centre for Social Research

Niall Harvey, ACM Global Laboratories

3.1. Introduction and key conclusions

This section describes the assay of analytes for the 2024 Scottish Health Survey (SHeS) biological samples and the quality control and quality assessment procedures that were carried out during the survey period. Details of procedures used in the collection, processing and transportation of the specimens are available on request from ScotCen Social Research.

The overall conclusion for the data provided in this chapter is that methods and equipment used for the measurement of saliva analytes produced internal quality control (IQC) within expected limits. The results of the analyses for the saliva cotinine and anabasine levels were acceptable for SHeS 2024.

3.2. Analysing laboratories

As in previous years, the saliva samples were initially sent to the Royal Victoria Infirmary (RVI) in Newcastle upon Tyne where they were checked for correct identification and stored prior to dispatch to ACM Global Laboratories in York. ACM conducted the analysis of salivary cotinine and anabasine.

3.3. Samples collected

A saliva sample was obtained from participants aged 16 and over. Saliva samples were collected for analysis of cotinine (a metabolite of nicotine that shows recent exposure to tobacco smoke) and anabasine (present in trace amounts in tobacco smoke, and can be used as an indicator of a person's exposure to tobacco smoke). A saliva collection tube was used for this purpose. Participants were also offered the option to provide the saliva sample using a dental roll that they could saturate with their saliva before it was placed in the tube. The saliva tube was then labelled and packaged ready for dispatch.

3.4. Methodology

Saliva samples received at the RVI were checked for correct identification, assigned a laboratory accession number, and stored at 4°C. Samples were checked for details and despatched fortnightly in polythene bags (20 samples per bag) by courier for overnight delivery to ACM Global Laboratories, where the cotinine and anabasine analysis was carried out. This laboratory specialises in accurate measurement of low levels of cotinine and anabasine and therefore takes special precautions to ensure no contamination by environmental tobacco smoke occurs. Additionally, control blanks are included in each analysis batch and assessed on a batch by batch basis Control blanks consist of blank surrogate saliva, interferences in these are quantified and are deemed acceptable if less than 20% of the lower limit of quantitation (LLOQ).

The method of analysis used since the 2009 SHeS study is high performance liquid chromatography coupled to tandem mass spectrometry with multiple reaction monitoring (LC-MS/MS), replacing the gas chromatography nitrogen phosphorous detection (GC-NPD) method used in earlier SHeS studies[1]. Prior to 2024 (excluding 2020 and 2021 when no samples were taken), analysis was undertaken using a three-stage method. Samples were divided for analysis into batches of self-reported smokers and non-smokers and analysed either using a method with a high calibration range, 1.00 to 750 ng/mL for the self-reported smokers, or low calibration range 0.100 to 50.0 ng/mL for the non-smokers If any of the samples from self-reported smokers gave a result below 1.00 ng/mL on initial analysis they were repeated in a low range batch. Similarly, if any of the non-smoker samples gave a result above 50 ng/mL then they were repeated in a high range batch. Samples from self-reported smokers are analysed for anabasine within the range 0.100 – 10.0 ng/mL. Sample from non-smokers that produced a cotinine concentration >10.0 ng/mL were then also analysed for anabasine. The high range cotinine method and anabasine method had a validated dilution factor of 5 enabling quantitation up to 2500 ng/mL and 40.0 ng/mL for the respective analytes.

A new single-stage method was developed for analysis of the 2024 samples. This method quantifies cotinine between 0.100 – 500 ng/mL and anabasine between 0.100 – 15.0 ng/mL, samples with a concentration above these ranges were re-assayed with a dilution. A 10-fold dilution was successfully validated enabling cotinine quantitation up to 2000 ng/mL and anabasine up to 60.0 ng/mL. This approach suitably covered the ranges quantified by the three previous methods.

All methods were validated before use.

3.5. Internal quality control (IQA)

ACM ran 8 calibration standards in duplicate (16 in total) for each batch of samples that were analysed. 6 quality control (QC) samples, two each at a set concentration to represent low, medium and high levels for the calibration range being used, were also analysed with each analytical batch. For the results from any analytical batch to be acceptable, four out of the six QCs must have a bias of no greater than ±20.0% with at least one from each QC level being within these acceptance criteria, and 75% of the calibration standards must have a bias of no greater than ±20% except at the lower limit of quantification where the bias must be no greater than ±25%. For batches including diluted samples, a dilution QC at a concentration above the analytical range was included in triplicate and diluted 10-fold prior to analysis, at least two of the three diluted QCs had to have a bias of no greater than ±20%.