The qualification of Cisplatin

The separation of Cisplatin is based upon the powerful technique: Hydrophilic interaction liquid chromatography (HILIC)
Sample preparation
Organic Hydrophobic Mobile phase eluent: DMF or acetonitrile, prepared from HPLC grade NH4OAc and Milli-Q water.
Column Hydrophilic stationary phase

Eluent: 1-propanol/Ammonium: 
Stock solution cis-platin: 1000 ppm= 1ug/mL, then to get standards the stock is diluted in different times.
Sample injection: about 30ul syringe, but the injection loop only 10 uL, be aware of air bubbles getting the syringe and loop.
Mass spectra. base the one the abundant times the M/z colliding to the detection table.
To calculate the concentration of cisplatin, the area of the peaks can be made a chart with the concentration.
The sample(cancer cells ) stored in the refrigerator, exposed to 20uM cisplatin,
The calibaration curve made from the concentrations of standards against the  peak areas of platinum.
. There are 2 isotopes of platinum.
The total platinum is determined following: run blank first using eluent, then  the 500uL(diluted 50 or 25 times of 10uL in 1-propanol) sample is directly injected to plasma(without passing the column). around 500uL is enough for one time measurement. 5 points from the intensity/time will be recorded. The running method is already set.
The remaining sample will be filtrated with membrane to keep bonds-cisplatin and cell. time 15min and temperature 15-20 degree Celsius, at speed 11000 prn. Then they will be injected to the LC-ICP-MS.
- The standards from 1ppb-3ppb-10ppb also injected for quality control.
- Also a series of standards from 0.5ppb to 2.5ppb cis-platin diluted from 1000ppm stock solution will be run LC-ICP MS, they are diluted in ammonium chloride to be kept stable.

First the cancer cell was taken out of the affected person. Then they were incubated with 20 uM cisplatin. stored in refrigerator. Then it was grown in the ''human invironment''. The incubation time is varied 0-5-10-15-20-30, then the sample is analyzed by LC-ICPMS