Fast, Simple LC-MS/MS Analysis of Creatine and Creatinine
Featured Application: Creatine and Creatinine in Plasma and Urine on Raptor HILIC-Si
- Simple isocratic method, no complex mobile phases.
- Fast, 1.5 min analysis without additional equilibration time.
- Highly reproducible results.
Creatine is a compound that is made primarily in the liver and then transported to muscles where it is used as an energy source. Once in the muscle, some of the creatine is spontaneously converted to creatinine. It is important to monitor creatine and creatinine because these compounds can be used as biomarkers of renal, liver, and heart health. Historically, colorimetric and enzymatic assays have been used, but LC-MS/MS is a better technique because it reduces interferences. Although LC-MS/MS analysis of creatine and creatinine using an alkyl-bonded (e.g., C18) column is the usual approach, these highly polar analytes are very difficult to retain and separate from each other using a reversed-phase column. When a reversed-phase method is employed, complex ion-pairing reagents (e.g., tetrabutylammonium hydroxide) are generally required to increase analyte hydrophobicity.
Using HILIC, ion-exchange, or other newer LC phases can be a better alternative to reversed-phase methods because these phases employ different mechanisms of interaction. Some non-C18 columns do provide increased retention, but analysis times can be long (e.g., 10 min) and may require the use of complex mobile phases and gradients. Labs needing faster LC-MS/MS analysis of creatine and creatinine should consider adopting the HILIC method shown here. Good separations in both plasma and urine samples were obtained in just 1.5 min using a Raptor HILIC-Si (2.7 μm, 50 mm × 2.1 mm) column and a simple, isocratic mobile phase. Results during in-house testing were highly reproducible, making this fast, simple LC-MS/MS analysis a good procedure for high-throughput labs.
Human Plasma
| Peaks | tR (min) | Precursor Ion | Product Ion | Product Ion | |
|---|---|---|---|---|---|
| 1. | Creatinine | 0.537 | 114.0 | 44.3 | 86.0 |
| 2. | Creatine | 1.232 | 132.1 | 43.3 | 90.2 |
| Column | Raptor HILIC-Si (cat.# 9310A52) | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Dimensions: | 50 mm x 2.1 mm ID | |||||||||
| Particle Size: | 2.7 µm | |||||||||
| Pore Size: | 90 Å | |||||||||
| Guard Column: | UltraShield UHPLC precolumn filter 0.2 µm (cat.# 25810) | |||||||||
| Temp.: | 40 °C | |||||||||
| Standard/Sample | ||||||||||
| Diluent: | 20:80 Water:acetonitrile | |||||||||
| Conc.: | Endogenous levels | |||||||||
| Inj. Vol.: | 0.2 µL | |||||||||
| Mobile Phase | ||||||||||
| A: | 5 mM Ammonium formate in 20:80 water:acetonitrile | |||||||||
|
| Detector | MS/MS |
|---|---|
| Ion Mode: | ESI+ |
| Mode: | MRM |
| Instrument | UHPLC |
| Sample Preparation | Endogenous levels of creatinine and creatine in human plasma were determined using a single protein precipitation step followed by LC-MS/MS analysis. A 50 μL aliquot of human plasma (K2EDTA) was mixed with 950 μL acetonitrile. After vortexing and centrifuging at 4300 rpm for 10 min, 200 μL of the supernatant was transferred to a new vial and mixed with 50 μL of water. Centrifugation was performed again before injection. |
| Notes | Endogenous peaks for phospholipids and lysophospholipids are displayed because they are common matrix interferences and are known to suppress ionization efficiency. |
Human Urine
| Peaks | tR (min) | Precursor Ion | Product Ion | Product Ion | |
|---|---|---|---|---|---|
| 1. | Creatinine | 0.537 | 114.0 | 44.3 | 86.0 |
| 2. | Creatine | 1.240 | 132.1 | 43.3 | 90.2 |
| Column | Raptor HILIC-Si (cat.# 9310A52) | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Dimensions: | 50 mm x 2.1 mm ID | |||||||||
| Particle Size: | 2.7 µm | |||||||||
| Pore Size: | 90 Å | |||||||||
| Guard Column: | UltraShield UHPLC precolumn filter 0.2 µm (cat.# 25810) | |||||||||
| Temp.: | 40 °C | |||||||||
| Standard/Sample | ||||||||||
| Diluent: | 20:80 Water:acetonitrile | |||||||||
| Conc.: | Endogenous levels | |||||||||
| Inj. Vol.: | 0.2 µL | |||||||||
| Mobile Phase | ||||||||||
| A: | 5 mM Ammonium formate in 20:80 water:acetonitrile | |||||||||
|
| Detector | MS/MS |
|---|---|
| Ion Mode: | ESI+ |
| Mode: | MRM |
| Instrument | UHPLC |
| Sample Preparation | Endogenous levels of creatinine and creatine in human urine were determined using a simple dilute-and-shoot method. A 50 μL aliquot of human urine was mixed with 950 μL acetonitrile. After vortexing and centrifuging at 4300 rpm for 10 min, 10 μL of the supernatant was added to the 1490 μL of 80% acetonitrile in water. Centrifugation was performed again before injection. |