Antibiotics protocols - Gibbons-Lab/wiki GitHub Wiki

Antibiotics HPLC quantification

These are the used protocols to prepare the stool samples for quantification of cefoperazone. Stocks are prepared in water and suspended in Acetonitrile. Note that Cefoperazone and Ceftiofur are not soluble in pure Acetonitrile.

sample list

Sample preparation mouse fecal samples

Prepare:

  • IS ceftiofur at 50 ug/mL in water
  • 10% water /90% acetonitrile (ACN)
  • 60% water/ 40% ACN
  1. Mouse stool sample, weigh back empty tube
  2. Transfer in Precellys vial
  3. Add 750ul solvent of 10% water /90% acetonitrile (ACN)
  4. Add 50ul internal standard ceftiofur at 50 ug/mL -> total solvent volume is 800ul
  5. Precellys, 2 cycles (cool 5min on ice inbetween?)
  6. Spin 8 min, 16,000 rpm, 4°C
  7. Remove supernatant (550ul? Don’t try to take everything, clear solution important)
  8. Mix/quick vortex before dilution, then dilute 1:250 with 60% water/ 40% CAN -> take 5ul sample + 1245 ul solvent -> freeze remaining sample in case we are too diluted for some samples
  9. Pipette 100ul in autosampler vial, freeze remaining samples
  10. Inject 5ul on column

Note: I suggest to do the precellys / centrifugation step in smaller sample batches and not 20 or 38 at a time, just in case one tube should break, then others do not get damaged, and I noticed the sample separate relatively fast, thus do step 7. Right after step.6 and avoid samples sitting too long.

IS concentration on column:

  • IS 50ug/mL = 0.05ug/ul
  • 50ul IS in total volume of 800ul -> (50ul0.05ug/ul)/800ul = 3.12510 -3 ug/ul
  • Dilute 1:250 (5+1245) and inject 5ul -> (3.125*10 -3 ug/ul : 250)5=6.2510 -5 ug on column

Blood Samples

  1. 50ul blood
  2. Add 500 ul acetonitrile
  3. Add 12ul water (Millipore)
  4. Add 38 ul IS ceftiofur at 0.4ug/mL (dissolved in water; this is the 50 ug/mL further diluted 1:125 (e.g., 5+620) -> total volume 600ul, water content approx. 16% (50+12+38 in 600 total)
  5. Vortex for 3min
  6. Centrifuge properly (high/make note) for 8 min at 4C
  7. Remove supernatant (make note how much. Don’t try to take everything, need particle free solution)
  8. Take 50ul supernatant and add 50ul Millipore water (1:1 dilution to get to~58% water to bind sample on column, 16% in 50ul SN =8ul; total 58 water in 100ul)
  9. Pipette 100ul in autosampler vial, freeze remaining samples
  10. Inject 5ul on column

IS concentration on column:

  • IS 50ug/mL = 0.05ug/ul
  • Dilute 1:125 -> 0.4*10 -3 ug/ul
  • 40ul IS in total volume of 600ul -> (38ul0.410 -3 ug/ul)/600ul = 25.3*10 -6 ug/ul
  • Dilute 1:1 (50+50) and inject 5ul -> (25.3*10 -6 ug/ul: 2)5=6.3310 -5 ug on column -> similar as fecal samples