You can try to:

Check the health and morphology of the cells under a microscope. Look for any signs of contamination, stress, or poor viability that could impact dopamine production.

Test a fresh vial of cells from an early passage to rule out genetic drift causing loss of dopamine phenotype.

Be sure the cells are being properly lysed and that dopamine is not being degraded during processing.

Run a spiked sample with added dopamine standard to rule out an issue with detection.

Check dopamine metabolizing enzymes - are they still expressing? Is dopamine being converted to downstream products before analysis?

Consider analyzing mRNA or protein levels of enzymes involved in dopamine synthesis to see if expression has decreased.

There are several factors that could contribute to the absence of dopamine peaks in your HPLC analysis of MN9D cells. Here are some suggestions to investigate:

1. Cell passage number: Although you mentioned that your passage numbers are low, it's possible that the cells are still undergoing senescence or accumulating mutations that affect their ability to produce dopamine. Try using younger cultures or passaging the cells again to refresh the population.

2. Culture conditions: Check that your culture conditions, such as media composition, pH, temperature, and oxygen levels, remain consistent and optimal for dopamine production. Ensure that the cells are exposed to sufficient amounts of glucose, fetal bovine serum, and other essential nutrients.

3. Dopamine synthesis pathway inhibitors: Verify that none of the reagents used in your culture or experimental procedures are inhibiting the dopamine synthesis pathway. For example, certain antibiotics like streptomycin can inhibit tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis.

4. Tyrosine availability: Confirm that the cells have access to enough tyrosine, the precursor to dopamine. Tyrosine starvation can lead to reduced dopamine production. Supplementing the medium with tyrosine might help.

5. Enzymatic degradation: Dopamine can be metabolized by various enzymes, such as monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT). Elevated activity of these enzymes could reduce the amount of detectable dopamine. Consider adding inhibitors for MAO (e.g., pargyline) or COMT (e.g., entacapone) to your assay to minimize their impact.

6. Protein expression: Assess whether TH protein expression levels are affected. Reduced TH expression or activity could result in lower dopamine production. Use western blot or immunocytochemistry techniques to evaluate TH protein levels in your MN9D cells.

7. Genetic alterations: It's possible that genetic drift or mutations have occurred in your MN9D cell line, leading to changes in dopamine production. Perform DNA sequencing to identify potential mutations in genes related to dopamine synthesis and transport.

8. HPLC parameters: Double-check your HPLC settings, such as column type, mobile phase composition, flow rate, and detection wavelength, to ensure they are optimized for dopamine detection. You may need to adjust the HPLC conditions to improve sensitivity or selectivity.

9. Sample handling and storage: Make sure sample handling and storage protocols are proper to avoid dopamine degradation or loss during processing. Minimize exposure time to light, oxygen, and contaminants. Keep samples cold (e.g., on ice or in a refrigerator) until HPLC analysis.

10. Reference standards: Verify the authenticity and purity of your dopamine reference standards. Contaminated or impure standards can give rise to aberrant results. Obtain new standards from a trusted supplier if needed.

11. Instrument maintenance: Regularly service and maintain your HPLC instrument to ensure its performance and accuracy.

Methodically addressing each of these points should help resolve the issue and restore the detection of dopamine in your MN9D cell HPLC analyses.

All the best