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A 5,000 ng/mL standard mix was prepared in urine and diluted with urine to make calibration standards at 2, 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, and 5,000 ng/mL (only continine and trans -3-hydroxycotinine were tested at 2,500 and 5,000 ng/mL levels)

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Gestational diabetes screening often uses different protocols that don't require overnight fasting