QUANTIFICATION OF DRUGS IN BIOLOGICAL SAMPLES
The disclosure provides methods for the detection and quantification of drugs and drug metabolites in biological samples, such as naloxone, buprenorphine, and norbuprenorphine.
1 . A method for quantifying naloxone, buprenorphine, and norbuprenorphine in a human plasma sample comprising:
a. obtaining about 0.1 ml to about 1.0 ml of a plasma sample from a human;
b. performing a liquid-liquid extraction technique on the plasma sample to produce a first composition comprising naloxone, buprenorphine, and norbuprenorphine;
c. combining the first composition with a mobile phase comprising ammonium bicarbonate and methanol to produce a second composition comprising ammonium bicarbonate, methanol, naloxone, buprenorphine, and norbuprenorphine;
d. performing high pressure liquid chromatography separation on the second composition using gradient elution to produce a third composition comprising naloxone, buprenorphine, and norbuprenorphine; and
e. performing electrospray ionization mass spectrometry on the third composition to determine the concentration of the buprenorphine, naloxone, and norbuprenorphine;
thereby quantifying naloxone, buprenorphine, and norbuprenorphine in the human plasma sample.
2 . A method for quantifying naloxone, buprenorphine, and norbuprenorphine in a biological sample comprising:
a. obtaining the biological sample
b. performing an extraction technique on the biological sample to produce a first composition comprising naloxone, buprenorphine, and norbuprenorphine;
c. combining the first composition with a mobile phase comprising an alcohol to produce a second composition comprising the alcohol, naloxone, buprenorphine, and norbuprenorphine;
d. performing liquid chromatography separation on the second composition using gradient elution to produce a third composition comprising naloxone, buprenorphine, and norbuprenorphine; and
e. performing electrospray ionization mass spectrometry on the third composition to determine the concentration of the buprenorphine, naloxone, and norbuprenorphine;
thereby quantifying naloxone, buprenorphine, and norbuprenorphine in the biological sample.
3 . The method of claim 1 or 2 , wherein the electrospray ionization mass spectrometry electrospray ionization mass spectrometry is tandem mass spectrometry.
4 . The method of claim 2 or 3 , wherein the liquid chromatography separation is high pressure liquid chromatography separation.
5 . The method of any one of claims 1 - 4 , wherein the biological sample is a human biological sample.
6 . The method of any one of claims 1 - 5 , wherein the biological sample is human serum, human plasma, human tissue, or human cells.
7 . The method of any one of claims 1 - 6 , wherein the biological sample is human plasma.
8 . The method of any of claims 2 - 7 , biological sample is in an amount of about 0.1 ml to about 1 ml.
9 . The method of any one of claims 1 - 8 , wherein the biological sample is in an amount of about 0.1 ml to about 0.5 ml.
10 . The method of any one of claims 1 - 9 , wherein the patient is a human.
11 . The method of any one of claims 1 - 10 , wherein the mobile phase has a varied flow rate.
12 . The method of any one of claims 1 - 11 , wherein the gradient elution has a schedule of:
Time
Mobile Phase A
Mobile Phase B
(min)
(%)
(%)
0.00
50
50
0.50
50
50
2.00
20
80
3.00
20
80
3.01
20
80
4.00
20
80
4.50
0
100
5.50
0
100
5.51
50
50
6.00
50
50
6.01
50
50
13 . The method of any one of claims 1 - 12 , wherein the gradient elution has a schedule and flow rate of:
Time
Mobile Phase A
Mobile Phase B
Flow Rate
(min)
(%)
(%)
(mL/min)
0.00
50
50
0.5
0.50
50
50
0.5
2.00
20
80
0.5
3.00
20
80
0.5
3.01
20
80
0.8
4.00
20
80
0.8
4.50
0
100
0.8
5.50
0
100
0.8
5.51
50
50
0.8
6.00
50
50
0.8
6.01
50
50
0.5
14 . The method of claim 12 or 13 , wherein Mobile Phase A comprises ammonium bicarbonate in water; and wherein Mobile Phase B comprises ammonium bicarbonate in methanol.
15 . The method of any one of claims 12 - 14 , wherein Mobile Phase A comprises about 0.5 mM to about 5 mM ammonium bicarbonate in water; and wherein Mobile Phase B comprises about 1 mM to about 10 mM ammonium bicarbonate in methanol.
16 . The method of any one of claims 2 - 15 , wherein the extraction technique is a solid-phase extraction technique.
17 . The method of any one of claims 1 - 15 , wherein the extraction technique is a liquid-liquid extraction technique.
18 . The method of claim 1 or 17 , wherein the liquid-liquid extraction comprises a C 2-6 linear or branched alkyl ether and a C 1-10 linear or branched alkane.
19 . The method of any one of claims 1 - 18 , wherein the mobile phase is alkali.
20 . The method of any of claims 1 - 19 , wherein the mobile phase comprises ammonium bicarbonate.
21 . The method of any of claims 1 - 20 , wherein the mobile phase comprises ammonium hydroxide.
22 . The method of any of claims 1 - 21 , wherein the mobile phase comprises ammonium bicarbonate in water.
23 . The method of any of claims 1 - 22 , wherein the mobile phase comprises ammonium bicarbonate in methanol.
24 . The method of any of claims 1 - 23 , wherein the mobile phase has a pH of about 5 to about 10.
25 . The method of any of claims 1 - 24 , wherein the mobile phase has a pH of about 6 to about 9.
26 . The method of any of any one of claims 1 - 25 , wherein the mobile phase has a pH of about 7.9.
27 . The method of any one of claims 1 - 26 , wherein quantifying naloxone, buprenorphine, and norbuprenorphine occurs simultaneously.