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Detection of N-in-1 Experiments: Extending the Range of N Using the API 3000 LC/MS/MS System


Increased throughput in drug development laboratories can be obtained using the API 3000 LC/MS/MS System for N-in-1 experiments. Patented LINAC collision cell technology plays a key role in maximizing throughput by enabling faster scan times without sacrificing sensitivity.


A significant area in which LC/MS/MS has made an important impact is in N-in-1 studies (also referred to as cassette dosing or cocktail dosing) typically performed in pre-clinical pharmaceutical analyses. In these studies, several new chemical entities (NCEs) are administered simultaneously to a single animal, and their pharmacokinetic (PK) behavior is subsequently characterized. The advantages of cassette dosing include higher throughput of NCEs and fewer numbers of animals used. This results in overall greater screening efficiency and faster data attainment. However, combined drug interactions are always a concern in multiple dosing studies since you may obtain potentially misleading PK results. One remedy is to decrease the individual dose of each NCE. This alternative is made possible by the sensitivity and specificity of LC/MS/MS techniques.

Improved in instrument performance now makes it possible to decrease the total analysis time for NCEs. It is now possible to simultaneously and confidently detect many compounds in a single sample. The N-in-1 approach accelerates the drug discovery process and also reduces the cost of candidate screening. The API 3000 triple quadrupole LC/MS/MS System offers superior sensitivity and selectivity for the detection of compounds even at very low levels.

Key Features

Patented LINAC collision cell allows for faster scan rates without compromising data quality

Analyst software provides fast and easy data acquisition and reduction

Proven Turb oIonSpray and APCI (Heated Nebulizer) ion source technologies give maximum performance across the widest range of flow rates and mobile phase compositions

Experimental Conditions

The sample set evaluated consists of dog plasma extracts collected at 4, 6, 8, 10, 12 and 24 hours after administration of seven compounds: A, B, C, D, E, F, and G. Extracted plasma samples were evaporated and reconstituted without internal standard in 200 μL of 25% acetonitrile: 75% water: 0.1% formic acid (v/v/v).

Initially, a 7-in-1 experiment was performed observing eight multiple reaction monitoring (MRM) transitions seven compounds of interest plus an internal standard. The molecular masses of all eight compounds ranged from 400 to 600 amu. After completing this initial run, the dwell time was reduced to improve throughput. To maintain the same number of points across each peak, the MRM transitions were increased to 40 by incorporating 33 dummy transitions, thereby essentially performing a 40-in-1 experiment.

An API 3000 LC/MS/MS instrument was used to analyze specimens containing these seven compounds sampled at the six collection times. All seven compounds were detected on the API 3000 system even 24 hours after administration. To further demonstrate the sensitivity improvement of the API 3000 LC/MS/MS System, samples were also analyzed on an API III+ LC/MS/MS. The weakest compound, Compound C, was last detected on the API III+ at 4 hours after drug administration; and the second weakest compound, Compound F, was last detected 10 hours after drug administration. On the API 3000 system, these two weaker compounds were still easily seen in specimens sampled 24 hours after doping.

During the 40-in-1 experiment, five of the seven compounds were detected at the 24 hour sampling. The two compounds present in the lowest concentrations, C and F, were last detect ed at 12 hours, which was still a significant improvement over the 7-in-1 experiment performed on the API III+. The remaining five compounds were all detected on the API 3000 LC/MS/MS System well above the LOD at the 24 hour sampling time.


These experiments demonstrate the feasibility of using shorter dwell times for simultaneous monitoring of multiple compounds. Faster scan rates were used, extending the 7-in-1 MRM experiment into a 40-in-1 MRM experiment without a significant loss in sensitivity. Using the API 3000 LC/MS/MS System for N-in-1 experiments can shorten the drug discovery timeline by increasing the number of compounds detected per sample.



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