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First, the bias covariates used, although carefully extracted and evaluated, were based on observable study characteristics and thus may not fully capture unobserved characteristics such as study quality or context, which might be major sources of bias

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The two phases separate after extraction and an aliquot of the aqueous layer (water/acetonitrile/acetate buffer extract) was spiked with internal standard, extracted, centrifuged and analysed as per the pouch samples