Need help with IS calibration calculation

[YYC-718]

I was wondering if someone could help me here..I got myself really confused with the extraction and IS calculation...Thank you in advance for your help!!

I recently followed a method to extract Vitamin D3 from my sample as follow--
5 g of sample + 0.2 mL of IS + 40 mL of pyrogallol alcohol + 80 mL of KOH, then extract into 80 mL of hexane. But only 40 mL of the hexane was evaporated to dryness and reconstituted in 2 mL of acetonitrile.

IS calibration standards-- 0.2 mL of IS was added into 2 mL of each calibration standards.

Calibration curve-- Y axis- area of D3/area of IS, x-axis- conc. of Vit D3 (mcg/L)

Calculation #1:
concentration (mcg/g) of Vit D in my sample = Conc. from calibration curve (mcg/L) * 0.002 L * (1/5 g)

or

Calculation #2:
concentration (mcg/g) of Vit D in my sample = conc. from calibration curve (mcg/L) * 0.002 L * (1/5g) *(80 mL/40 mL)

Which calculation is correct? Calculation #1 or calculation #2?

[dblux_]

If you are determining vitamin D3 and you are adding vitamin D3 to samples and standards then this is not an internal standard calibration.

[YYC-718]

my IS is trideuterated D3.

[dblux_]

I was wondering if someone could help me here..I got myself really confused with the extraction and IS calculation...Thank you in advance for your help!!

I recently followed a method to extract Vitamin D3 from my sample as follow--
5 g of sample + 0.2 mL of IS + 40 mL of pyrogallol alcohol + 80 mL of KOH, then extract into 80 mL of hexane. But only 40 mL of the hexane was evaporated to dryness and reconstituted in 2 mL of acetonitrile.

IS calibration standards-- 0.2 mL of IS was added into 2 mL of each calibration standards.

Calibration curve-- Y axis- area of D3/area of IS, x-axis- conc. of Vit D3 (mcg/L)

Calculation #1:
concentration (mcg/g) of Vit D in my sample = Conc. from calibration curve (mcg/L) * 0.002 L * (1/5 g)

or

Calculation #2:
concentration (mcg/g) of Vit D in my sample = conc. from calibration curve (mcg/L) * 0.002 L * (1/5g) *(80 mL/40 mL)

Which calculation is correct? Calculation #1 or calculation #2?

Calculation #1 - provided your calibration is correct (I don't want to get into details but when "IS calibration standards-- 0.2 mL of IS was added into 2 mL of each calibration standards." then you get 2,2 mL)